Keaney, 2000 (Atherosclerosis From Lesion Formation To Plaque)

Molecular Aspects of Medicine 21 (2000) 99166
www.elsevier.com/locate/mam
Atherosclerosis: from lesion formation to plaque

activation and endothelial dysfunction
1
John F. Keaney Jr.
Department of Medicine and Pharmacology, Whitaker Cardiovascular Institute,
Boston University School of Medicine, 715 Albany Street, Room W507, Boston, MA 02118, USA
Abstract
Atherosclerosis is an important source of morbidity and mortality in the developed world.
Despite the fact that the association between LDL cholesterol and atherosclerosis has been
evident for at least three decades, our understanding of exactly how LDL precipitates ath-
erosclerosis is still in its infancy. At least three working hypotheses of atherosclerosis are now
nearing the stage where their critical evaluation is possible through a combination of basic
science investigation and murine models of atherosclerosis. As we move forward in our un-
derstanding of this disease, eorts will be increasingly focused on the molecular mechanisms of
disease activation that precipitate the clinical manifestations of atherosclerosis such as heart
attack and stroke. Two candidates for such investigation involve the events surrounding
plaque activation and endothelial dysfunction. Further investigation in these elds should
provide the necessary insight to develop the next generation of interventions that will reduce
the clinical manifestations of this devastating disease. The purpose of this work is to review the
major theories of atherogenesis, examine the aspects of atherosclerosis that lead to disease
activation and discuss aspects of disease activation that are amenable to treatment. 2000
Published by Elsevier Science Ltd.
Keywords: Atherosclerosis; Inammation; Endothelium; Nitric oxide; Oxidation
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
2. Atherogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
2.1. Epidemiology and risk factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
2.2. Morphologic features of atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
2.2.1. The normal artery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
2.2.2. Gross morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
1
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100 J.F. Keaney Jr. / Molecular Aspects of Medicine 21 (2000) 99166
2.3. Response-to-injury hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

2.4. Oxidative modication hypothesis of atherosclerosis . . . . . . . . . . . . . . . . . . 107
2.4.1. LDL does not support foam cell formation . . . . . . . . . . . . . . . . . . . . 107
2.4.2. Mechanisms of LDL oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
2.4.2.1. Structure of LDL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
2.4.2.2. Stages of LDL oxidation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
2.4.2.3. Oxidation of LDL in cell-free systems . . . . . . . . . . . . . . . . . . . . . 112
2.4.2.4. Metal ion-induced LDL oxidation . . . . . . . . . . . . . . . . . . . . . . . 113
2.4.2.5. Superoxide and LDL oxidation . . . . . . . . . . . . . . . . . . . . . . . . . 114
2.4.2.6. Lipoxygenase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
2.4.2.7. Glycoxidation reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
2.4.2.8. Peroxynitrite . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
2.4.2.9. Myeloperoxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
2.4.3. Proatherogenic actions of oxidized LDL . . . . . . . . . . . . . . . . . . . . . . . 116
2.4.4. Evidence to support the oxidative modication hypothesis . . . . . . . . . . 118
2.4.5. Antioxidant studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
2.4.6. Animal studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
2.4.6.1. Vitamin E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
2.4.6.2. Probucol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
2.4.7. Problems with the oxidative modication hypothesis . . . . . . . . . . . . . . 122
2.4.8. Clinical studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
2.4.9. Evidence from descriptive studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
2.4.9.1. Evidence from case control studies . . . . . . . . . . . . . . . . . . . . . . 125
2.4.9.2. Evidence from prospective cohort studies . . . . . . . . . . . . . . . . . . 125
2.4.9.3. Randomized trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
2.5. The response-to-retention hypothesis of early atherogenesis . . . . . . . . . . . . . 126
2.5.1. Evidence to support the response-to-retention hypothesis . . . . . . . . . . . 126
3. The concept of disease activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
3.1. The brous cap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
3.2. Matrix degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
4. Vascular homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
4.1. Oxidative stress and impaired NO bioactivity . . . . . . . . . . . . . . . . . . . . . . . 134
4.1.1. Superoxide and NO bioactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
4.1.2. Lipid peroxidation and NO bioactivity . . . . . . . . . . . . . . . . . . . . . . . . 137
4.1.3. Lipid-soluble antioxidants and NO bioactivity . . . . . . . . . . . . . . . . . . 137
4.1.3.1. a-Tocopherol and b-Carotene . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
4.1.3.2. Probucol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
4.1.4. Water-soluble antioxidants and NO bioactivity . . . . . . . . . . . . . . . . . . 140
4.1.4.1. Glutathione . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
4.1.4.2. Ascorbic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
4.1.5. Other means of treating endothelial dysfunction . . . . . . . . . . . . . . . . . 144
4.1.5.1. L -arginine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
4.1.5.2. Tetrahydrobiopterin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
4.1.5.3. LDL lowering therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
4.1.5.4. Other treatments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
5. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146
J.F. Keaney Jr. / Molecular Aspects of Medicine 21 (2000) 99166 101
1. Introduction
Cardiovascular disease is the major source of morbidity and mortality in the

Western World. In 1997, the latest year for which statistics are available, cardio-
vascular disease claimed 953,110 lives in the United States where presenting 41% of
all deaths (American Heart Association, 2000). The magnitude of this problem is
simply staggering, cardiovascular disease claims more lives each year than the next
seven leading causes of death combined. Approximately 1/6 all of people killed by
cardiovascular disease are under the age of 65, and the estimated age-adjusted
prevalence of cardiovascular disease is 30% for men, and 24% for women (American
Heart Association, 2000). In the black population, this number climbs to 41% for
men, and 40% for women (American Heart Association, 2000). Using the United
States economic costs, cardiovascular disease consumes in excess of 20 billion dollars
in direct healthcare costs, and if one includes lost productivity, the cost climbs to
over 250 billion dollars annually (American Heart Association, 2000). Although one
might be tempted to believe this is principally a problem of the developed world, the
World Health Organization predicts that continued global economic prosperity will
likely lead to an epidemic of cardiovascular disease as developing countries continue
acquire Western habits. Clearly then, cardiovascular disease is a major public health
problem that will only have wider implications over the ensuing decades.
The principal manifestations of cardiovascular disease are heart attack and
stroke; these represent the clinical sequelae of a systemic vascular process known as
atherosclerosis. Data developed from epidemiological studies over the last 40 yrs has
shed light on the predilection of cardiovascular disease for the Western world. These
ndings relate to the fact that atherosclerosis is a disease of aging, and that pre-
mature atherosclerosis can be precipitated by a number of clinical conditions, the
most prominent of which include excess LDL cholesterol, diabetes mellitus, hyper-
tension, and cigarette smoking (Dawber et al., 1957; Kannel and McGee, 1979).
Another major risk factor for premature atherosclerosis is a family history of pre-
mature atherosclerosis in a rst degree relative. The denition of premature ath-
erosclerosis includes a rst-degree male relative with clinical evidence of
cardiovascular at an age <55, or a rst-degree female relative with clinical mani-
festations of cardiovascular disease prior to age 60. Of these risk factors for car-
diovascular disease, excess LDL cholesterol has received the most attention of late.
This is perhaps tting as it was among the rst of the risk factors identied for
cardiovascular disease and remains the principal target today for modifying future
risk of cardiovascular disease with cholesterol lowering agents.
Atherosclerosis is an insidious process that may persist for many years before
clinical manifestations become evident. This observation is due to the fact that the
processes involved in atherogenesis (i.e., the process of early lesion development)
require prolonged exposure to predisposing factors. It is only the latter stages of
disease that progress rather rapidly and lead to clinical manifestations. The processes
of atherosclerosis lesion development and the clinical events of atherosclerosis are
distinct. Due to this distinction, therapies that are eective at preventing lesion de-
velopment may not prove to be eective in preventing clinical manifestations of the
disease. The principal reason is that many therapies are instituted after individuals
have established lesions. Indeed, since most individuals initiate atherosclerosis before
they are teenagers (Stary, 1983), it would be very dicult to devise a strategy to
prevent lesion development. For these reasons, this review will discuss the processes
of lesion development, and the clinical manifestations of atherosclerosis separately.
Where possible, overlapping mechanisms will be identied and discussed.
2. Atherogenesis
2.1. Epidemiology and risk factors
At the outset, one should understand that atherogenesis is a multifactorial

process the complexity of which makes it dicult to clearly dene the risk attrib-
utable to any one risk factor. To be dened as a risk factor, a trait must be causally
linked to a particular disease in a manner that is strongly and consistently associ-
ated with that disease. Because most risk factors do not occur in a vacuum, the
apparent strength of the association will depend on whether or not analyses have
control for other concurrent conditions that might contribute to the disease. One
good example illustrating the need to control for associated variables is obesity.
Obesity is clearly associated with an increased risk of atherosclerosis, however, this
association seems to be mediated in large part by abnormalities in diabetes, hy-
pertension, and lipoproteins; all known risk factors for atherosclerosis. The purpose
of this discussion is not to provide a primer in epidemiology. Rather, this section
will focus on established risk factors and their contribution to the development of
atherosclerosis.
Cigarette smoking. The link between cigarette smoking and heart disease dates
back to the 1940s and 1950s when a series of studies unequivocally linked smoking
and heart disease (English et al., 1940; Doll and Hill, 1956; Hammond and Horn,
1958). More recently, the Surgeon General's report estimates that smoking increases
atherosclerotic disease by more than 50% and doubles the incidence of coronary
heart disease (US Department of Health and Human Services, 1989). This excess risk
of smoking on coronary disease is readily reduced through smoking cessation. In
fact, the risk of heart attack in ex-smokers declines to almost that of non-smokers
over two years (Gaziano, 1996).
Hypertension. Hypertension is dened as a systolic blood pressure in excess of 140
mm Hg or a diastolic blood pressure above 90 mm Hg (Joint National Committee on
Detection, 1993). Current estimates indicate that hypertension is more prevalent
among blacks than whites, and among men than women, with approximately 30% of
the American population qualifying as hypertensive. The elderly also appear to have
an increased predilection to hypertension with up to 75% of people over 75 yrs of age
qualifying for the risk factor (Joint National Committee on Detection, 1993). There
appears to be an approximately linear relation blood pressure elevation and the
increased incidence of atherosclerotic vascular disease with an increase of 7 mm Hg
and diastolic blood pressure corresponding to a 27% increase in myocardial
infarction, and 42% increase in stroke (MacMahon et al., 1990). Antihypertensive

therapy has proven most eective in reducing stroke, with a 56 mm mercury re-
duction in blood pressure corresponding to a 42% reduction in the risk of stroke, but
only a 14% reduction in the risk of myocardial infarction (Collins et al., 2000).
Diabetes mellitus. Atherosclerotic coronary disease is a major complication of
diabetes mellitus, a diagnosis that encompasses approximately 14,000,000 people in
the United States. In patients with diabetes the risk of coronary atherosclerosis is 35
fold greater than non-diabetics despite controlling for other risk factors (Bierman,
1992; Pyorala et al., 1987). A number of other known risk factors for coronary
disease such as hypertension and abnormal lipids are also more common in diabetics
than the general population (Bierman, 1992), but despite this association, no more
than 25% of the excess coronary atherosclerosis risk from diabetes can be attributed
to these known risk factors (Nishigaki et al., 1981). Thus, diabetes represents a major
contributing factor to atherosclerosis in the developed world and much of this excess
risk has escaped quantication using traditional risk factor analysis. Although im-
proved glucose control in diabetes is associated with a reduction in a number of
diabetic complications, coronary atherosclerosis is not reproducibly among these
complications reduced by improved diabetic control.
Serum cholesterol. The association between cholesterol and atherosclerosis is
unequivocal. Among the strongest evidence for this association is an experiment of
nature. Familial hypercholesterolemia is an autosomal dominant disorder that af-
fects approximately one in 500 persons from the general population. Heterozygotes
for this disease manifest a 23 fold elevation in plasma cholesterol that is solely due
to an elevation in LDL. The molecular basis for this disease is now well understood
and relates to functional defects in the LDL receptor that interfere with LDL
clearance. Homozygotes for this disorder demonstrate a 46 fold elevation in plasma
cholesterol. One of the major features of FH disease is precocious atherosclerosis. In
heterozygotes, 85% of individuals have experienced a myocardial infarction by the
age of 60, and this age is reduced to 15 in patients who are homozygous for the
disease. A similar defect has been reported in rabbits that is also associated with
precocious atherosclerosis.
With respect to patients in general, approximately 50% of all Americans between
the ages of 20 and 74 have cholesterol levels that exceed 200 mg/dl (Lehr et al., 1999).
The excess risk of increased LDL cholesterol appears to be linear with a 1% increase
in serum cholesterol corresponding to a 2% increase in the risk of coronary heart
disease. This risk can be lowered greatly by cholesterol lowering therapy, and with
the advent in the last 10 yrs of HMG CoA reductase inhibitors. There has been a
dramatic improvement in the morbidity and mortality from cardiovascular disease in
patients with hypercholesterolemia.
Since most models of atherosclerosis employ LDL cholesterol as a central feature,
the remainder of this review will be written from the standpoint that LDL cholesterol
is a necessary feature of atherosclerosis. While this may not be true in all respects, it
serves as a reasonable working hypothesis to use as a framework for discussion of
the known features of both atherosclerosis and the clinical manifestations of coro-
nary vascular disease.
2.2. Morphologic features of atherosclerosis
2.2.1. The normal artery

The arterial wall (Fig. 1) normally consists of three well-dened concentric layers:
the innermost layer is called the intima, the middle layer is called the media, and the
outermost layer is known as the adventitia. These three layers are demarcated by
concentric layers of elastin known as the internal elastic lamina (which separates the
intima from the media), and the external elastic lamina (which separates the media
from the adventitia).
The luminal surface of arteries is lined by a single contiguous layer of endothelial
cells that sits upon a basement membrane of extracellular matrix, and is bordered by
the internal elastic lamina. Endothelial cells are attached to one another by a series of
interconnections known as junctional complexes. The amount of extracellular matrix
and elastin in the internal elastic lamina is most prominent in medium and large-
sized arteries. The endothelial cells form a dynamic barrier between the luminal
surface of the artery and the stroma of the arterial wall. At one time it was thought
that endothelial cells were quite passive in their function, but it is now known that
endothelial cells regulate a wide array of functions in the arterial wall including
thrombosis, vascular tone, and leukocyte tracking within the arterial wall.
Progressing outwards from the internal elastic lamina, the media consists also of a
single cell type, in this case, smooth muscle cells. There can be one or many layers of
smooth muscle cells depending on the size of the artery. Cells are held together by an
extracellular matrix comprised largely of elastic bers and collagen, and cells may
also be attached together by junctional complexes. The extracellular matrix within
the arterial wall is produced predominantly by the smooth muscle cell. This includes
collagen, proteoglycans, and elastic bers.
Fig. 1. Cross-section of a normal artery.

The media can vary in size considerably based on the function of a given artery.
For example, in very small arteries the media may only be one cell thick, or not
present at all, whereas in large arteries like the aorta the media may be many cell
layers thick and consist of considerable elastin as part of the extracellular matrix.
The principal reason for this dierence relates to the requirement for elastic recoil
during diastole, the period when the heart is not beating.
Beyond the external elastic lamina, the adventitia is the outermost layer of the
artery. The adventitia typically consists of a loose matrix of elastin, smooth muscle
cells, broblasts, and collagen. Most of the neural input into blood vessels also
traverses the adventitia. At one time, the adventitia was considered quite passive in
nature, but contemporary thinking now includes adventitial broblasts in the arterial
response to insult.
2.2.2. Gross morphology

Atherosclerosis typically is manifested in three stages known as early, developing,
and mature lesions. Early lesions are characterized by nodular areas of lipid depo-
sition that have been termed ``fatty streaks'' morphologically. These represent lipid-
lled macrophages and smooth muscle cells in focal areas of the intima. Fat-soluble
dyes readily demonstrate these areas for gross morphologic examination and the
principal lipid stained by these fat-soluble dyes is cholesteryl oleate. These early
lesions typically develop by age 10, and increase to occupy as much as 1/3 of the
aortic surface in the third decade.
Developing lesions are sometimes called pearly plaques and represent the next
stage beyond fatty streaks. They can be found initially in areas of the coronary
arteries, abdominal aorta, and some aspects of the carotid arteries in the third to
fourth decade of life. These brous plaques are dome-shaped and rm, and are
covered by a bromuscular layer known as a ``cap''.
Ultimately, lesions may progress to become complicated and advanced, and these
are characterized by calcied brous areas of the artery with visible ulceration. These
are the types of lesions that are often associated with symptoms or arterial embo-
lization. It was once thought that end-organ damage and infarction was due to
gradual advancement of these lesions, but we now know a number of functional
components in the arterial wall are also important determinants of end-organ
damage.
2.3. Response-to-injury hypothesis
Based on autopsy studies, it had been known for some time that thrombosis and
lipids were involved in atherosclerosis. On of the earliest theories involving throm-
bosis was the `ìncrustation'' theory of Rokitansky (1852) that suggested intimal
thickening results from brin deposition in the arterial wall. The lipid component of
atherosclerosis was encompassed by a competing contemporary hypothesis oered
by Virchow in 1858 (Virchow, 1989) who proposed that lipid transudation into the
arterial wall and complexation with mucopolysaccharides was the initial event
in atherosclerosis. Principally as a function of his work on smooth muscle cell
proliferation, Ross eventually proposed the ``response to injury'' hypothesis of

atherosclerosis (Ross et al., 1977). The conceptualization of atherosclerosis as an
injurious event to the arterial wall really brought to bear the idea that the devel-
opment of lesions and the progression of atherosclerotic disease were a dynamic
event rather than one characterized by senescence.
In 1973, Ross and Glomset (1973) attempted to reconcile a number of observa-
tions into a unifying hypothesis they termed the ``response-to-injury''. In this hy-
pothesis, they proposed that the initial step in atherogenesis was endothelial
denudation leading to a number of compensatory responses that alter the normal
vascular homeostatic properties. For example, this injury increases the adhesiveness
of the endothelium for leukocytes and platelets, and tips the local environment from
an anticoagulant milieu to a procoagulant one. The recruited leukocytes and
platelets release a number of cytokines, vasoactive agents, and growth factors setting
the stage for an inammatory response. This inammatory response is characterized
by migration and proliferation of smooth muscle cells from the media that become
admixed with the inammatory area to form an intermediate lesion.
The macrophages recruited to the arterial wall take up deposited LDL lipid to
form lipid-laden macrophages also known as foam cells. This is the hallmark of an
early atherosclerotic lesion. The accumulation of lipid-laden foam cells perpetuates
an inammatory response leading to a localized collection in the arterial wall not
unlike an abscess that would be observed in other tissues. This space-occupying
lesion may encroach upon the arterial lumen, but the artery can compensate up to a
point such that the arterial lumen remains unaltered, the so-called Glagov eect
(Glagov et al., 1987). The principal mediators of this early inammatory response
are macrophages and tea lymphocytes (Jonasson et al., 1986; van der Wal et al.,
1989).
Within this localized lesion continued inammation can lead to cellular necrosis
and further recruitment of monocytes and lymphocytes with a concomitant release
of cytokines, growth factors, and proteolytic enzymes. This can set the stage for focal
necrosis within the lesion, and can cause autocatalytic expansion of the lesion. As the
lesion enlarges the artery can no longer compensate for the encroachment in the
lumen, and at some point ow is impaired.
The response-to-injury hypothesis was originally based on the notion of endo-
thelium desquamation as a principal event initiating atherosclerosis (Ross and
Glomset, 1976). More recently however, it has become clear that developing ath-
erosclerotic lesions are covered by an intact endothelial cell layer, and that endo-
thelial desquamation is the exception rather than the rule. In a recent renement to
his initial hypothesis, Ross proposed that endothelial dysfunction is sucient to
initiate atherogenesis through increased endothelial permeability to atherogenic
lipoproteins (Ross, 1999). This hypothesis is not without its problems. It is clear that
even in normal artery segments, the rate of LDL entry into the arterial wall exceeds
the rate of accumulation (Carew et al., 1984), suggesting that endothelial dysfunction
is not strictly necessary for atherogenic lipoprotein entry into the arterial wall. In
fact, the accumulation of atherogenic lipoproteins in the arterial wall appears to be
concentrated in areas that are predisposed to future lesion development even though
the rate of entry is similar to normal sites (Schwenke and Zilversmit, 1989a,b). These
studies and several others (Schwenke and Carew, 1989a,b; Falcone et al., 1984) in-
dicate that lesion-prone arterial sites show an enhanced retention of atherogenic
lipoproteins containing apo B. Such shortcomings in the response-to-injury hy-
pothesis have prompted alternative hypothesis for the initiation of atherosclerosis
that will be discussed below.
2.4. Oxidative modication hypothesis of atherosclerosis
2.4.1. LDL does not support foam cell formation

Since macrophages were identied as the predominant cell type given rise to foam
cells, the hallmark of early lesions, it is not surprising that an intensive eort was
undertaken to investigate the mechanisms of foam cell formation. Despite the un-
deniable association of elevated LDL cholesterol levels in atherosclerosis, it is in-
teresting that LDL particles in and of themselves do not appear to be atherogenic in
vitro. Initially investigators anticipated that foam cell formation was mediated by the
LDL receptor. However, this assumption turned out to be incorrect. One clue to the
problem with this assumption is derived from patients with FH disease. These pa-
tients lack functional LDL receptors; nevertheless, they manifest precocious ath-
erosclerosis as early as the rst decade of life. Therefore, it seems unlikely that a
functional LDL receptor in and of itself is required for foam cell formation.
Moreover, incubation of LDL with normal macrophages possessing normal LDL
receptors does not support foam cell formation (Goldstein et al., 1979a). In fact, high
concentrations of LDL generally lead to down-regulation of the LDL receptor as
receptor-mediated endocytosis is tightly regulated (Goldstein et al., 1979a). In
searching for alternative LDL receptors, Brown and Goldstein observed that
chemical modication of LDL in the form of acetylation leads to foam cell forma-
tion when incubated with macrophages. The uptake of this chemically modied LDL
was shown to take place by way of a saturatable, specic receptor later termed the
`àcetyl-LDL receptor'' (Goldstein et al., 1979a). This receptor is now one of many
so-called ``scavenger receptors'' that are present on macrophages and other cell types
(Krieger et al., 1993).
In their original paper, Brown and Goldstein (Goldstein et al., 1979a) could not
identify any known endogenous means of LDL acetylation in vivo. They did,
however, speculate that other modications of LDL as yet undiscovered may fa-
cilitate recognition of LDL by the acetyl-LDL receptor. This prediction proved
prescient as Henriksen and colleagues (Henriksen et al., 1981) found that incubation
of LDL with endothelial cells modied LDL in such a way that it served as a ligand
for foam cell formation and macrophages. It is now well established that there is a
large family of so-called ``scavenger'' receptors present on macrophages and other
cells (Krieger, 1997). The original acetyl-LDL receptor has since been cloned and is
now known to exist in two forms termed scavenger receptor A1 and A2 (Kodama
et al., 1990). Many other scavenger receptors have also been identied including
CD68, CD36, SR-B1, LOX-1, among others (Krieger, 1997). Considerable evidence
does suggest that these receptors play a role in atherosclerosis. Mice lacking the SRA gene
demonstrate a defect in the binding and degradation of modied LDL (Krieger,

1997). In order to investigate the role of SRA receptors in atherosclerosis, these mice
were crossed into a well-established mouse model of atherosclerosis, namely mice
lacking the apolipoprotein E gene. Mice lacking the apo E gene typically demon-
strate atherosclerosis when placed on a high fat diet (Suzuki et al., 1997). However,
when these mice also lack the SRA receptor, the extent of atherosclerosis is signif-
icantly inhibited (Suzuki et al., 1997). Similarly, macrophages derived from mice
lacking the CD36 scavenger receptor demonstrate reduced uptake of modied LDL
(Nozaki et al., 1995) and reduced atherosclerosis when crossed with the apo E null
mouse (Febbraio et al., 2000). Thus, scavenger receptors appear to play an impor-
tant role in the development of atherosclerosis. It remains to be seen what the precise
contribution of each class of scavenger receptors and whether genetic variations in
these receptors have any implications for the development of atherosclerosis and its
clinical events in a patient population. One obvious question with the discovery of
the family of scavenger receptors is their precise role that favored their persistence
throughout evolution. This appears to involve the recognition and disposal of a
variety of modied biomolecules.
The current theory of the oxidative modication hypothesis states that LDL
becomes oxidized in the arterial wall where it then lends itself to cellular uptake and
foam cell formation. Of critical importance to this hypothesis is the mechanism of
LDL oxidation both in vitro and in vivo. We know considerably more about the
former than the latter, and in the ensuing pages we will discuss what is known about
the mechanisms of LDL oxidation.
2.4.2. Mechanisms of LDL oxidation

2.4.2.1. Structure of LDL. The precise characterization of LDL oxidation has been
problematic. One, this is largely a function of both the complexity and heterogeneity
of human LDL both amongst individuals and in response to dietary variation.
Human LDL is a particle containing both lipid and protein that is typically isolated
by ultracentrifugation between the densities of 1.019 and 1.063 g/ml (Gotto and
Farmer, 1988). Physically, LDL is spherical with a diameter that ranges between 19
and 25 nm, and a molecular weight between 1.8 and 2.8 million (Keaney and Frei,
1994). The lipid composition of LDL is outlined in Table 1. Given an assumed
molecular weight of approximately 2.5 million, an LDL particle consists of a lipo-
philic core containing approximately 1600 molecules of cholesteryl ester, and 170
molecules of triglyceride (Esterbauer et al., 1990). The LDL particle surface is also
embraced by a single apolipoprotein B-100 (apo B). Apo B is a glycosylated with
approximately 4500 amino acids corresponding to a molecular weight of 550,000
kDa (Gotto and Farmer, 1988).
With respect to the lipid classes within an LDL particle, a prototypical particle
contains 2700 fatty acid molecules (Esterbauer et al., 1990; Yla-Herttuala et al.,
1998), about half of these fatty acids are polyunsaturated fatty acids (PUFAs) with
the predominant PUFAs being linoleic acid (18:2) and arachidonic acid (20:4), and a
relatively minor quantity of docosahexaenoic acid (22:6) Table 1 (Esterbauer et al.,
Table 1
Lipid composition of human LDLa
nmol/mg LDL protein mol/mol LDL
Mean S.D. Mean
Total phospholipids 1300 227 700 122
Phosphatidylcholine 818 143 450 78
Lysophosphatidylcholine 145 25 80 14
Sphingomyelin 336 59 185 32
Fatty acids 2700
Linoleic acid 2000 541 1100 298
Palmitic acid 1260 375 693 206
Palmitoleic acid 80 44 44 24
Stearic acid 260 118 143 65
Oleic acid 825 298 454 164
Arachidonic acid 278 100 153 55
Docosahexaenoic acid 53 31 29 17
Free fatty acids 48 26
Triglycerides 304 140 170 78
Free cholesterol 1130 82 600 44
Total cholesterol 4090 2200
Conjugated dienes Not detectable
a
Taken from Keaney and Frei (1994).
1987). The content of PUFAs can vary considerably amongst LDL populations on
an individual basis with some reports demonstrating that the content of linoleic acid
may vary as much as 100% (Esterbauer et al., 1990). The importance of this fact is
emphasized by the knowledge that LDL lipid peroxidation is generally restricted to
PUFAs and therefore, LDL samples may vary considerably with respect to their
susceptibility for oxidative modication.
Another important characteristic of LDL-associated antioxidants (Table 2) is
that, by nature, they are lipid soluble. The most abundant lipid soluble antioxidant
in LDL by far is a-tocopherol with approximately 68 molecules per particle. All of
the other lipid soluble antioxidants are presents at amounts <1 molecule per LDL
particle. These include c-tocopherol, uniquinol-10, b-carotene, lycopene, crypto-
xanthine, and a-carotene (Table 2). Of these lipid soluble compounds, it appears
Table 2
Antioxidant content of human LDLa
Antioxidant nmol/mg LDL protein mol/mol LDL
Mean S.D. Mean
Vitamin E (a c-tocopherol) 15.5 2.9 7.95
Ubiquinol-10 0.65 0.28 0.33
b-carotene 0.53 0.47 0.27
Lycopene 0.41 0.20 0.21
Cryptoxanthine 0.25 0.23 0.13
a-carotene 0.22 0.25 0.11
a
Values are derived from Esterbauer et al. (1992), Frei and Gaziano (1993).
only that a-tocopherol and ubiquinol-10 possess important antioxidant activity

within the LDL particle. Since most of these compounds are contained in the diet,
one might expect the content of lipid soluble antioxidants will vary considerably
among individuals as a function of several factors including diet and the rate of fat
absorption.
2.4.2.2. Stages of LDL oxidation. The oxidative modication of LDL has been ar-
bitrarily divided into three stages. The rst is known as the initiation of lipid per-
oxidation and involves the initial formation of radical species within the particle. The
second stage is known as the propagation stage of oxidation and represents the
portion of LDL oxidation involving a chain reaction (i.e., each radical produced in
the particle yields more that one subsequent radical). The nal stage of LDL oxi-
dation is known as decomposition predominantly because lipid hydroperoxides
formed within the LDL particle decompose into reactive aldehydes and ketones.
This leads the modication of the apo B moiety of LDL and changes the net charge
of LDL. This division of LDL oxidation into stages is quite arbitrary and used for
conceptual purposes only. The actual oxidation process is a continuum that is not
readily divided into such distinct stages.
A stylized scheme of lipid peroxidation is contained in Fig. 2. The earliest event in
the modication is the initiation of lipid peroxidation shown in the gure as ab-
straction of hydrogen from a PUFA. In this case the oxidant is hydroxyl radical, an
extremely potent free radical with a very high reactivity and short half-life (109 s).
The predilection for an oxidant attacking a hydrogen bound to a carbon that is
anked by two double bonds is based on the carbon hydrogen bond energy. The
hydrogen shown in Fig. 1 is termed a bis-allylic methylene group and the anking
double bonds reduce the carbon hydrogen bond energy to between 75 and 70 kcal/
mol (Wagner et al., 1994). Despite all our knowledge about lipid peroxidation, the
precise entity responsible for the initiation of lipid peroxidation in biologic systems is
not known. A number of species have been proposed that include hydroxyl radical,
Fe2 =Fe3 =O2 (Minotti and Aust, 1987), peroxynitrite, tyrosyl radical, and even
enzyme systems such as lipoxygenase. This question of initiation is extremely im-
portant since LDL isolated from plasma is generally free of pre-formed of lipid
hydroperoxides based on the most sensitive techniques (Sattler et al., 1994).
Once lipid hydroperoxides have been established in the LDL particle, it is rela-
tively easy to generate radical species especially in the presence of metal ions as
shown in Eqs. (1) and (2).
LOOH Men1 ! LOO Men H 1
Men LOOH ! LO OH Men1 2

In lipid systems that contain trace amount or preformed lipid hydroperoxides
(LOOH), metal ions (Me) such as copper or iron can catalyze the decomposition of
lipid hydroperoxides into peroxyl LOO and alkoxyl LO radicals as shown
above. These peroxyl and alkoxyl radicals readily react with other adjacent bis-allylic
Fig. 2. Scheme for lipid peroxidation. In this scheme, hydroxyl radical initiates lipid peroxidation through
the abstraction of hydrogen (A) from a bis-allylic methylene group in a polyunsaturated fatty acid
(PUFA). The carbon-centered radical so formed undergoes molecular rearrangement to form a conjugated
diene compound exhibiting UV absorbance at 234 nm (B). The carbon-centered radical then reacts readily
with molecular oxygen to form a lipid peroxyl radical (C) that may then propagate lipid peroxidation
through the abstraction of hydrogen from an adjacent PUFA forming both a lipid hydroperoxide and
another carbon-centered radical (D), the lipid peroxyl radical.
methylene groups (LH) by extracting hydrogen atoms and forming lipid hydroper-
oxides and lipid hydroxides, as well as carbon-centered radicals by the scheme
outlined below
LOO LH ! LOOH L 3
LO LH ! LOH L 4
L O2 ! LOO 5
This decomposition of lipid hydroperoxides and the secondary generation of alkoxyl
and peroxyl radicals has been termed re-initiation of LDL lipid peroxidation (Gokce
and Frei, 1996) since the existence of pre-formed hydroperoxides within the LDL
particle indicates prior oxidative events (van der Wal et al., 1989). Whatever the
mechanism, the introduction of a free radical into a lipoprotein particle by nature
must generate a chain reaction. The reason for this is quite straight forward, free
radicals contain unpaired electrons whereas non-radical species (such as those in an
LDL particle) are spin-paired. The product of such a reaction must, invariably,
contain an uneven number of electrons and result in the production of a free radical.
This radical product must then react with another non-radical species, and by this
mechanism, a single radical will produce damage to a number of subsequent PUFAs

and hence the term ``chain reaction''. If one traces the sequence outlined in Fig. 2,
one can see that the carbon-centered radical rapidly takes up oxygen to produce a
lipid peroxyl radical (C) that may abstract a hydrogen from an adjacent PUFA
regenerating a lipid hydroperoxide and another carbon-centered radical to begin the
cycle of events again (Fig. 1). This chain reaction portion of LDL oxidation takes
place during the propagation phase, which is characterized by the consumption of
polyunsaturated fatty acids (PUFAs) and the formation of lipid hydroperoxides
within the LDL particle.
Lipid hydroperoxides, in particular phospholipid hydroperoxides, formed
within the LDL particle can be hydrolyzed to lysophospholipids, and fatty acid
hydroperoxides by a phospholipase A2 activity that has is intrinsic to apo B (Partha-
sarathy and Barnett, 1990), or a platelet activating factor (PAF) acetylhydrolase-
like activity that also appears to be associated with LDL (Steinbrecher and
Pritchard, 1989). Such decomposed hydroperoxides can undergo beta-scission
reactions that form aldehyde compounds, and those measured during this process
include 4-hydroxynonenal, malondialdehyde, and 2,4-heptadienal (Esterbauer
et al., 1987). Aldehydes have a predilection for amino groups and readily form Schi
bases with the lysine groups in apo B (Haberland et al., 1984; Steinbrecher, 1987).
The net result of these reactions is to change the charge of apo B, as lysine residues
are positively charged at physiologic PH and their charge is lost during the for-
mation of a Schi base. This is manifested as an increased electrophoretic mobility
of LDL upon electrophoresis, and this change in charge is important with respect
to LDL receptor recognition. In particular, modied apo B with its increased net
negative charge is no longer recognized by the apo B/E LDL receptor (Brown and
Goldstein, 1986) and instead recognized by any number of these ``scavenger
receptors'' is in macrophages (Goldstein et al., 1979a; Sparrow et al., 1989; Free-
man et al., 1991).
Recognition by the scavenger family of receptor is essential for the formation of
foam cells. As discussed above, receptor-mediated endocytosis of native LDL is
tightly regulated whereas scavenger receptor-mediated LDL uptake is not. Uptake of
modied LDL by the scavenger receptor pathway is at least 310 fold more ecient
than native LDL uptake (Henriksen et al., 1983).
2.4.2.3. Oxidation of LDL in cell-free systems. With its relatively high content of
PUFAs, LDL oxidation does occur spontaneously (Gurd, 1960), but also requires
many months of low temperatures (Lee, 1980). Early investigations into the oxida-
tion of LDL involved the relative ecacy of a number of metal ions in producing
LDL oxidation (Ray et al., 1954) with the most eective agent identied being
copper (Ray et al., 1954; Nichols et al., 1961), and the demonstration that metal ion
chelators such as ethylenediaminetetraacetic acid (EDTA) eectively inhibited LDL
oxidation (Ray et al., 1954; Schuh et al., 1978). Subsequent investigation demon-
strated that the protein moiety of LDL also underwent modication initially
described as degradation (Schuh et al., 1978).
2.4.2.4. Metal ion-induced LDL oxidation. The precise mechanisms involved in metal
ion-induced LDL oxidation have been dicult to elucidate. As shown in Fig. 2, lipid
peroxidation is initiated by the abstraction of a bis-allylic hydrogen atom from LDL
PUFAs. With respect to metals, there is no chemical evidence that copper or iron are
capable of abstracting hydrogen atoms directly, and initiated lipid peroxidation
(Halliwell and Gutteridge, 1990). One proposed mechanism to get around this
problem has been the participation of copper or iron in the generation of hydroxyl
radical from hydrogen peroxide via Fenton chemistry, as shown below.
2O
2 2H ! H2 O2 O2 6
H2 O2 Men1 ! OH OH Men 7
O
2 Me
n
! O2 Men1 8
However, there are several problems with this model. The equations outlined above
demonstrate that superoxide is a critical component of this model and consistent
with this assumption, SOD has been shown to inhibit copper-mediated LDL oxi-
dation in vitro (Lynch and Frei, 1993; Parthasarathy et al., 1989). However, careful
examination reveals that this eect may be due to non-specic binding of copper ions
to SOD in a redox-inactive form rather than dismutation of superoxide (Jessup et al.,
1993). Another problem with the model relates to observations that catalase does not
appear to inhibit iron- or copper-induced LDL oxidation (Lynch and Frei, 1993), yet
hydrogen peroxide is required for the Fenton reaction (Hammond and Horn, 1958)
depicted above. Finally, ecient scavengers of aqueous hydroxyl radicals appear
ineective in inhibiting metal ion-induced LDL oxidation (Lynch and Frei, 1993).
One alternative to the initiation of lipid peroxidation by hydroxyl has been the
metal catalyzed breakdown of pre-formed lipid hydroperoxides as described in Eqs.
(1) and (2). However, the existence of pre-formed lipid hydroperoxides in LDL is
controversial. While some investigators claim hydroperoxides are present in vivo
(Esterbauer et al., 1992), many of these ndings could be due to the method of LDL
isolation. In fact, rapid isolation of LDL from plasma produces lipoproteins that
contain no detectable hydroperoxides (Sattler et al., 1994), whereas traditional
prolonged methods of LDL isolation produce abundant quantities of lipid hydro-
peroxides in LDL (Shwaery et al., 1998). Furthermore, it appears that pre-formed
hydroperoxides are not necessary for lipid peroxidation as removal of all hydro-
peroxides in LDL with the synthetic peroxidase ebselen generates hydroperoxide-free
LDL that is still susceptible to copper- and iron-mediated oxidation (Lynch and
Frei, 1993).
Once the initiation of lipid peroxidation is established, propagation of lipid per-
oxidation is readily accomplished by copper or iron as outlined above in Eqs. (1) and
(2). This process appears to be quite dependent upon the relative distribution of
oxidized versus reduced transition metal ions (Minotti and Aust, 1987). For exam-
ple, incubation of LDL with Cu2 is associated with its rapid reduction to Cu1 while
co-incubation of LDL with Fe3 does not yield as ecient a reduction of iron (Lynch
and Frei, 1995). In fact, LDL appears to have considerable capacity for the reduc-
tion of copper, and one might wonder where all the reducing activity comes from.
This quandary was addressed recently when it was found that a-tocopherol serves as
a reductant for copper in human lipoproteins and is responsible for triggering the
initiation of LDL oxidation (Kontush et al., 1996).
One potential problem with metal ion-induced LDL oxidation is the fact that the
existence of extracellular free metal ions or their low-molecular-weight chelates has
not been denitively established. Redox-active metal ions have been found in ho-
mogenates of atherosclerotic tissue (Smith et al., 1992; Swain and Gutteridge, 1995;
Lamb et al., 1995), but comparable treatment of normal tissue has not been exam-
ined. Low concentrations of albumin also inhibit metal ion-dependent LDL oxida-
tion (Thomas, 1992), and albumin is the most abundant protein in plasma. Thus, one
cannot be condent that the requirements for Fenton chemistry can be met in the
extracellular space of the arterial wall.
2.4.2.5. Superoxide and LDL oxidation. Superoxide is the one electron reduction
product of molecular oxygen and its production has been implicated in LDL lipid
peroxidation in vivo (Lynch et al., 1997). Inammatory cells produce superoxide as a
means of aiding the host defense mechanism (Klebano, 1980) and the biochemical
basis for superoxide production by these cells is well understood. Activation of in-
ammatory cells is associated with NADPH oxidase activity that catalyzes the direct
reduction of molecular oxygen to superoxide. Monocyte activation is known to
promote LDL oxidation (Hiramatsu et al., 1987; Cathcart et al., 1989) and this
reaction is prevented in the presence of superoxide scavengers. Most importantly,
LDL oxidation by inammatory cells appears to require superoxide production as
cells defective in this feature are not able to promote LDL oxidation (Hiramatsu
et al., 1987).
Superoxide is implicated in LDL oxidation by other cells as well. In fact, cell-
mediated oxidation of LDL has been demonstrated by all the major cell types in the
vascular wall including endothelial cells (Henriksen et al., 1981) and smooth muscle
cells (Morel et al., 1984; Heinecke et al., 1984). Evidence supporting superoxide and
cell-mediate LDL oxidation is derived from observations that it is inhibited by
superoxide dismutase (Heinecke et al., 1986; Stenbrecher, 1988) and that superoxide
generated enzymatically (Lynch and Frei, 1993), or by radiolysis (Bedwell et al.,
1989) also promotes LDL oxidation in the presence of metal ions. However, these
studies suer from many of the same drawbacks as metal ion-mediated LDL oxi-
dation since cell-mediated oxidation is almost uniformly inhibited in the presence of
metal ion chelators. Thus, there is considerable doubt as to whether superoxide plays
a role in oxidizing LDL in vivo consistent with the notion that superoxide at neutral
PH has an absolute requirement for metal ions in the promotion of LDL oxidation
(Lynch and Frei, 1993).
2.4.2.6. Lipoxygenase. The lipoxygenases are intracellular enzymes that add oxygen
to polyunsaturated fatty acids (Yamamoto, 1992). These enzymes are present in all
the major cell types of the arterial wall and have been observed in association with
atherosclerotic lesions (Yamamoto, 1992; Witztum and Steinberg, 1991; Berliner and
Heinecke, 1996). These ndings have fueled speculation that lipoxygenases may play
an important role in the oxidation of LDL that leads to atherosclerosis.
Such speculation has been bolstered by ndings that a combination of soy bean
lipoxygenase and phospholipases oxidize LDL in vitro (Sparrow et al., 1988).
Consistent with this speculation, lipoxygenase inhibitors prevented LDL oxidation
by cultured cells (Parthasarathy et al., 1989) although these ndings are not without
problems. For example, a number of lipoxygenase inhibitors are not specic, and
they also block metal ion-mediated LDL oxidation suggesting they have signicant
antioxidant properties (Jessup et al., 1991; Sparrow and Olszewski, 1991). The
hypothesis that lipoxygenases contribute to LDL oxidation has recently been
bolstered by observations that disruption of the lipoxygenase gene in mice dimin-
ishes atherosclerosis (Cyrus et al., 1999).
Not withstanding this enthusiasm, one must question how an intracellular enzyme
such as lipoxygenase facilitates LDL oxidation which is predominantly an extra-
cellular process. Among the proposed mechanisms for this phenomenon include the
idea that lipoxygenases produce lipid hydroperoxides that are then transferred into
LDL thereby ``seeding'' LDL and facilitating re-initiation of lipid peroxidation as
described above (Parthasarathy et al., 1989). If this hypothesis were correct, one
would expect to nd lipoxygenase products in early atherosclerotic lesions, and this
indeed is the case. The oxidation of LDL in vitro with 15-lipoxygenase exhibits a
ratio of S/R stereoisomers for 13-hydroxyoctadecanoic acid (13-HODE) that is
approximately 2.5/1. As expected, free radical-mediated LDL oxidation produces a
ratio that approaches 1. In two studies examining lipids from atherosclerotic pla-
ques, the S/R ratio of 13-HODE was 1.12 in advanced atherosclerotic lesions (Folcik
et al., 1995), and 1.08 in early atherosclerotic lesions (Kuhn et al., 1997). These small,
but statistically apparent increases in stereospecic 15-lipoxygenase products are
consist with the participation of 15-lipoxygenase in LDL oxidation.
2.4.2.7. Glycoxidation reactions. As discussed above, diabetes is a potent risk factor

for premature atherosclerosis. The increased risk of atherosclerosis associated with
diabetes has not been well identied, but one proposed mechanism includes excess
glucose since hyperglycemia is a hallmark of diabetes. This so-called ``glucose hy-
pothesis'' suggests that oxidation reactions induced by glucose help modify LDL to
forms that support the initiation of atherosclerosis and lesion development. This
hypothesis draws some support from studies showing that intensive glycemic control
in type I diabetics produces reduced vascular complications (Kuhn et al., 1990).
The molecular mechanism of glucose-mediated biological oxidation was rst
identied in 1912 by Maillard (1919). The Maillard reaction accounts for the glu-
cose-dependent non-enzymatic modication of proteins that accompanies hyper-
glycemia. Initially, this reaction involves the combination of the aldehyde group of
glucose (present in the open-chain form) with amine groups in proteins to form a
Schi Base followed by Amadori rearrangement to form the product fructoselysine.
This process is reversible and underlies the formation of hemoglobin A1C , a well-
recognized marker of chronic glycemic control. The persistence of the fructoselysine
produce may yield irreversible oxidation, or glycoxidation of fructoselysine to yield a

host of products which have been termed in aggregate, advanced glycation end-
products (AGEs). These irreversible oxidation products have been found to correlate
with the vascular and renal complications of diabetes, and thus represent an at-
tractive source of oxidation to explain precocious atherosclerosis in diabetes
(McCance et al., 1993). Although this may explain one mechanism for enhanced
atherosclerosis in diabetes, it does not provide an adequate explanation for LDL
modication in atherogenesis in non-diabetic subjects. For this reason, other sources
of oxidative stress capable of producing LDL oxidation are necessary.
2.4.2.8. Peroxynitrite. Peroxynitrite is a well-characterized product of the radical

radical combination reaction of superoxide and nitric oxide (Beckman et al., 1990).
Peroxynitrite is a two-electron oxidant that oxidizes LDL lipids and converts LDL
into a form that is recognized by macrophage scavenger receptors (Graham et al.,
1993). The possibility that peroxynitrite or other reactive nitrogen species might
support LDL oxidation during atherosclerosis has been examined in some detail.
Atherosclerotic lesions contain detectable levels of 3-nitrotyrosine (a marker of
peroxynitrite-mediated oxidation) that are 80-fold higher that in circulating LDL
(Leeuwenburgh et al., 1997). This observation is consistent with the notion that
peroxynitrite participates in the formation of oxidized LDL in the arterial wall.
2.4.2.9. Myeloperoxidase. In the host-defense response, phagocytes produce super-

oxide in such high concentrations that spontaneous dismutation into hydrogen
peroxide is signicant (Fridovich, 1983). One target of this hydrogen peroxide is the
phagocyte-secreted heme protein myeloperoxidase, which interacts with hydrogen
peroxide to generate a host of antimicrobial species (Klebano, 1980; Hurst and
Barette, 1989). Active myeloperoxidase has been observed in human atherosclerotic
lesions (Daugherty et al., 1994), and colocalizes with inammatory cells. In vitro,
myeloperoxidase readily oxidizes LDL, perhaps through the production of tyrosyl
radical (Savenkova et al., 1994). Among the markers for tyrosyl radical formation is
di-tyrosine, and it appears that tyrosyl radical may play a role in LDL oxidation
in vivo since dityrosine in detected in LDL isolated from atherosclerotic lesions
(Leuwenburgh et al., 1997).
Other important products of myeloperoxidase are hypochlorous acid (HOCl) and
chlorine gas. These oxidants produces chlorinated biomolecules, and therefore create
relatively specic markers for their oxidative damage. 3-chlorotyrosine is formed
in vitro in LDL incubated with myeloperoxidase-peroxide-chloride system (Hazen
et al., 1996). If one isolates LDL from human atherosclerotic tissue, 3-chlorotyrosine
is also readily detected suggesting a role for myeloperoxidase and these oxidants
in the modication of LDL (Hazen and Heinecke, 1997).
2.4.3. Proatherogenic actions of oxidized LDL

In addition to its activity to support foam cell formation, oxidized LDL (oxLDL)
also has a number of other proatherogenic properties that are outlined in Table 3.
Among the early events of atherosclerosis is endothelial cell activation, and products
Table 3
Potential proatherogenic activities of oxidized LDL (OxLDL)a
OxLDL has supports macrophage foam cell formation
OxLDL-derived products are chemotactic for monocytes and T-cells and chemostatic for tissue
macrophages
OxLDL-derived products are cytotoxic and can induce apoptosis
OxLDL is mitogenic for smooth muscle cells and macrophages
OxLDL can alter inammatory gene expression of vascular cells
OxLDL can increase expression of macrophage scavenger receptors
OxLDL can induce expression and activate PPARc , thereby inuencing many gene functions
OxLDL is immunogenic and elicits autoantibody formation and activated T-cells
Oxidation renders LDL more susceptible to aggregation, which independently leads to enhanced
uptake.
OxLDL is a substrate for sphingomyelinase, which aggregates LDL
OxLDL may enhance procoagulant pathways by induction of tissue factor and platelet aggregation
Products of OxLDL can aversely impact arterial vasomotor properties
a
Modied from Tsimikas and Witztum (2000).
of oxLDL have been shown to facilitate this activation (Kume and Gimbrone, 1994;
Khan et al., 1995). Hypercholesterolemia is a cardinal feature for experimental
models of atherosclerosis, and it is associated with increased LDL entry into the
arterial wall and reduced egress (Schwenke and Carew, 1989a,b). This is thought to
be due to increased LDL retention within the arterial wall (see the response-to-re-
tention hypothesis), and it is thought LDL undergoes oxidation while retained
within the arterial wall. Once oxidized, LDL increases its substrate suitability for
sphingomyelinase, an enzyme that is known to aggregate LDL and thereby enhance
its uptake by macrophages (Williams and Tabas, 1998). Initial oxidation of LDL
within the arterial wall forms a minimally modied form of LDL (MM-LDL) that
has a number of properties in and of itself. For example, MM-LDL stimulates ad-
jacent endothelial cells and smooth muscle cells to synthesize and secrete monocyte
chemotactic protein-1 (MCP-1) (Cushing et al., 1990). This local production of
MCP-1 (Navab et al., 1991) as well as the expression of endothelial leukocyte ad-
hesion molecules (Cybulsky and Gimbrone, 1991), are responsible for the recruit-
ment of monocytes that undergo activation-dierentiation in the subendothelial
space to become macrophages. A role for MCP-1 in the recruitment of inammatory
cells is supported by the isolation of MCP-1 protein and mRNA from macrophage
rich regions of human and rabbit atherosclerotic aorta (Yla-Herttuala et al., 1989).
Moreover, evidence consistent with a requirement for MCP-1 in atherogenesis is
derived from studies indicating that mice lacking the MCP-1-receptor demonstrate
decreased lesion formation in two murine models of atherosclerosis (Boring et al.,
1998; Gosling et al., 1999). This chemokine-mediated co-localization of monocytes
and LDL in the subendothelial space provides a facile environment for more
extensive LDL modication.
The formation of oxidized LDL in the subendothelial space also facilitates the
progression of atherosclerosis through other mechanisms. OxLDL is chemotactic for
monocytes (Quinn et al., 1987) and T lymphocytes (McMurray et al., 1993), perhaps
through LDL accumulation of lysophosphatidylcholine during oxidation (Steinbre-

cher et al., 1984). Macrophages exposed to oxLDL demonstrate impaired mobility
(Quinn et al., 1987) suggesting that oxLDL could prevent their egress from the arterial
wall. Furthermore, the accumulation of oxidized LDL and its by-products within the
arterial wall would be expected to produce impaired bioactivity of endothelium-
derived nitric oxide (Kugiyama et al., 1990) that could, conceivably enhance endothelial
permeability (Kubes and Granger, 1992) and stimulate local accumulation of LDL
and monocytes (Kubes et al., 1991). OxLDL has also been shown to be immunogenic
by eliciting the production of auto-antibodies (Parums et al., 1990; Salonen et al.,
1992) and the formation of immune complexes that can also facilitate macrophage
internalization of LDL (Klimov et al., 1985; Grith et al., 1988).
The gene expression pattern in the arterial wall is also subject to inuence by
modied forms of LDL. For example, MM-LDL as well as oxLDL can induce
macrophage scavenger receptor expression thereby enhancing foam cell formation
and LDL uptake (Mietus-Snyder et al., 1997). A number of genes associated with
inammation are also upregulated by oxLDL such as SAA, ceruloplasmin, and
heme oxygenase (Liao et al., 1993). In addition, a recent discovery is the eect of
oxLDL on macrophage expression of peroxisome proliferator PPARc expression.
This has been shown to alter scavenger receptor (CD36) expression and alter the
expression of pro-inammatory genes (Nicholson et al., 2000).
2.4.4. Evidence to support the oxidative modication hypothesis

Initially, there was considerable skepticism about the idea that LDL oxidation
could occur in vivo. This skepticism stems from the knowledge that numerous an-
tioxidants are present in plasma and extracellular uid that prevent oxidation of
LDL (Keaney and Frei, 1994; Dabbagh and Frei, 1995). There now are numerous
lines of evidence to indicate that LDL oxidation does occur in vivo. For example,
one can generate antibodies against the protein component of oxLDL. In particular,
aldehyde-modied epsilon amino groups of lysine such as malondialdehyde-lysine
(MDA-lysine), or 4-hydroxynonenal-lysine (4-HNE-lysine) have been generated and
used as a tool to determine if such epitopes exist in atherosclerotic lesions. Using this
approach, a number of investigators have demonstrated that antibodies raised
against such epitopes react with components of atherosclerotic lesions, and do not
react with normal arterial segments (Palinski et al., 1989; Haberland et al., 1988;
Palinski et al., 1990; Palinski et al., 1994; Hulten et al., 1996). With respect to human
studies, compared to normal age-match controls, patients with coronary athero-
sclerosis have elevated levels of endogenous antibodies to oxLDL (Salonen et al.,
1992). Furthermore, levels of plasma oxLDL are increased in patients who have
suered an acute myocardial infarction compared with age-matched controls (Hol-
voet et al., 1995).
The oxidation-specic epitopes discussed above are specic for the adduct (i.e.,
MDA-lysine or 4-HNE-lysine), but not for their existence in LDL per se. One could
argue that the immunological evidence presented above actually corresponds to
these epitopes on proteins other than apo B. To address this issue, Yla-Herttuala and
colleagues extracted LDL from atherosclerotic lesions of humans and experimental
animals (Yla-Herttuala et al., 1989). The compositional analysis of this LDL sug-
gested that it closely resembled LDL produced ex vivo with copper or other oxi-
dants. Most importantly, LDL isolated from these lesions demonstrated rapid
uptake by the macrophage scavenger receptors in a manner that competed eectively
for LDL that had been oxidized ex vivo (Yl a-Herttuala et al., 1989). Finally, if one
examines atherosclerotic lesions directly for the presence of oxidized lipids, one can
nd evidence of both lipid peroxidation (Suarna et al., 1995) and protein modi-
cation (Leeuwenburgh et al., 1997) that are similar to those observed during ex vivo
oxidation of LDL. Thus, there is clear evidence that LDL oxidation occurs in vivo.
A more pressing question, however, is whether this oxidation of LDL is a prereq-
uisite for atherosclerotic lesion formation and the progression of atherosclerosis.
2.4.5. Antioxidant studies

Principally as a consequence of the oxidative modication hypothesis, a number
of studies have been performed to examine the antioxidant protection of LDL and
ultimately, to apply this knowledge to animal studies in an attempt to inhibit ath-
erosclerosis. Table 4 contains selected small-scale clinical trials of antioxidant sup-
plementation in humans and its eect on ex vivo LDL oxidation. As one can
appreciate, the bulk of the trials have been done with vitamin E as it is the major
lipid-soluble antioxidant in human plasma and lipoproteins. In general, all of the
trials have demonstrated that oral consumption of vitamin E results in an increased
resistance of LDL particles to ex vivo oxidation. This eect appears to be observed
with a number of doses (Dieber-Rotherneder et al., 1991). Although not shown in
the table, most of the studies involving b-carotene alone did not demonstrate any
particular eect of b-carotene to increase the resistance of LDL particles to oxidation
(Reaven et al., 1993). Other lipid soluble antioxidants such as ubiquinol 10 (Mohr
et al., 1992) and probucol (Reaven et al., 1992; Cristol et al., 1992) have demonstrated
increased LDL resistance to oxidation in patients treated with these compounds.
Thus, there is considerable precedent that oral supplementation with lipid-soluble
antioxidants increases LDL particle resistance to oxidation. With this in mind, let us
examine animal studies undertaken to examine the eect of antioxidant supple-
mentation on atherosclerosis.
2.4.6. Animal studies

2.4.6.1. Vitamin E. Among the most obvious lipid soluble antioxidants to be studied
for the prevention of atherosclerosis is vitamin E. Table 5 contains selected studies of
vitamin E on the inhibition of atherosclerosis in experimental animals. It is inter-
esting to note that many earlier studies of vitamin E supplementation and its eect
on atherosclerosis predate awareness of the oxidative modication hypothesis
(Morgulis et al., 1938; Stamler et al., 1954; Beeler et al., 1962). Another feature of
these earlier studies is a cholesterol-lowering eect of vitamin E in these experimental
animals (Brattsand, 1975; Wilson et al., 1978; Westrope et al., 1982; Prasad and
Kalra, 1993). This is particularly important as cholesterol reduction with vitamin E
has produced a reduction in atherosclerosis, however, one cannot be convinced that
Table 4
Selected human studies of antioxidant supplementation and LDL oxidationa
Antioxidant(s) Dose Weeks N LDL levels Oxidative Eect on References
stress LDL
oxidation
Vitamin C 1.5 g/d 4 4 Smoking + Harats et al.
(1990)
Vitamin E 600 mg/d 4
a-tocopherol 150 IU/d 3 2 " 54% Cu2 + Dieber-Roth-
erneder et al.
(1991)
225 IU/d 2 " 106% +
800 IU/d 2 " 53% +
1200 IU/d 2 " 141% +
a-tocopherol 800 IU/d 12 12 Cu2 + Jialal and
Grundy (1992)
a-tocopherol 1600 mg/d 20 7 " 2.5-fold Cu2 , EC + Reaven et al.
(1993)
(AT)
b-carotene 60 mg/d 12 8 " 19-fold Cu2 , EC )
(BC)
a-tocopherol+ 1600 mg/d 12 8 AT " 1.3-fold Cu2 , EC +
b-carotene +60 mg/d BC " 22-fold
Vitamin C+ 2 g/d+ 8 8 Vitamin C n.d. Cu2 , EC +
a-tocopherol+ 1600 mg/d AT " 1.3-fold
b-carotene +60 mg/d BC " 37-fold
Vitamin C+ 900 mg/d+ 24 22 Cu2 + Abbey et al.
(1993)
a-tocopherol+ 200 mg/d+
b-carotene 18 mg/d
Ubiquinol-10 300 mg/d 11d 3 " 4-fold AAPH + Mohr et al.
(1992)
Probucol 250 mg/d 24 11 6 lg/mg Cu2 , EC + Reaven et al.
protein (1992)
1 g/d 7 12 lg/mg
protein
Probucol 250 mg/d 16 26 Cu2 + Cristol et al.
(1992)
a
Cu2 refers to copper ions in solution; AAPH refers to 2,2-azobis (2-amidinopropane) hydrochloride,
an aqueous peroxyl radical generator; EC refers to endothelial cells in culture. N refers to number of
subjects.
this is due to any of the antioxidant activity of vitamin E. This can be seen in Table 5,
with the exception of the mouse studies, a large number of the latter studies did not
demonstrate any material eect of vitamin E to reduce atherosclerosis. Studies in the
transgenic mice have generally been positive for an eect of vitamin E to inhibit
atherosclerosis although a few more studies will be required to see if this is a uni-
versal feature. The precise reasons behind such discrepant observations are not clear.
Possible explanations included material dierences in atherosclerosis between
models, as well as altered antioxidant metabolism in these two species. Further study
will be required to sort out such dierences.
Table 5
Selected studies of vitamin E and atherosclerosisa
Study Model Vitamin E dose % Change compared
to no supplement
Serum/plasma Lesion
cholesterol area
Williams et al. (1992b) Modied WHHL rabbits 5000 mg/kg diet )38 )32
Prasad and Kalra (1993) Cholesterol-fed rabbits 40 mg/kg/d )23 )73
Willingham et al. (1993) WHHL rabbits 2000 mg/kg diet )19 0
Kleinveld et al. (1994) WHHL rabbits 250 mg/kg diet 0 0
Fruebis et al. (1995) WHHL rabbits 1000 mg/kg diet 0 0
Shaish et al. (1995) Cholesterol-fed rabbits 100 mg/kg diet 0 0
Kleinveld et al. (1995) Oleate-fed WHHL rabbits 250 mg/kg diet 0 0
Linoleate-fed WHHL rabbits 250 mg/kg diet 0 0
Fruebis et al. (1997) WHHL rabbits 1000 mg/kg diet 0 0
Schwenke and Behr (1998) Cholesterol-fed rabbits 1375 mg/kg diet )17 0
Crawford et al. (1998) Cholesterol-fed 1000 mg/kg diet 0 )60
LDLR-decient micej
Pratico et al. (1998) Apo E ()/)) mice 2000 mg/kg chow 0 )66
a
All values for changes in cholesterol and lesion area are at study termination unless otherwise indicated.
The interpretation of animal antioxidant supplementation studies is not entirely

straightforward. Many of these studies suer from a lack of standardization as
control diets amongst studies are not comparable. In particular, the tocopherol
content of ``standard'' laboratory chows can vary quite considerably. This may have
major implications on experimental ndings as reported in the literature (Lehr et al.,
1999; Willy et al., 1995).
2.4.6.2. Probucol. Probucol is a cholesterol-lowering drug that is known to possess

considerable antioxidant activity (Marshall, 1982; Parthasarathy et al., 1986). Early
studies performed with probucol demonstrated that it inhibited the development of
atherosclerosis in rabbits (Kritchevsky et al., 1971; Tawara et al., 1986), as well as
monkeys (Wissler et al., 1983). Subsequent to these studies, probucol became the test
compound for the oxidative modication hypothesis (Kita et al., 1987; Carew et al.,
1987). Kita and colleagues (Kita et al., 1987) treated Watanabe Heritable Hyper-
lipidemic (WHHL) rabbits with probucol and observed an 87% reduction in lesion
area compared to those animals not treated with probucol. In addition, lipoproteins
derived from probucol-treated rabbits were much more resistant to copper-mediated
oxidation than LDL particles derived from the control animals (Kita et al., 1987).
The authors concluded the reduction of atherosclerosis was due to an antioxidant
eect, however, the treated group did demonstrate a 17% reduction in total cho-
lesterol compared to the control group, complicating the analysis. This issue was
dealt with in a subsequent study by Carew et al. (1987) in which the cholesterol-
lowering eect of probucol was oset by another treatment group with lovastatin, an
HMG-CoA reductase inhibitor that lowers cholesterol without antioxidant protec-
tion. Both lovastatin and probucol lowered the total cholesterol in WHHL rabbits
similarly, however, probucol treatment provided an additional 48% reduction in the

extent of atherosclerosis compared to lovastatin (Carew et al., 1987). These data
were taken a strong evidence that probucol inhibits atherosclerosis independent of
cholesterol-lowering with the idea that this additional eect was directly related to its
antioxidant activity.
A number of other studies have been conducted which also support the notion
that probucol reduces atherosclerosis, perhaps, through its antioxidant activity. In
one such study, WHHL rabbits were treated with a structural analog of probucol
that had no cholesterol-lowering properties (Mao et al., 1991). Compared to pro-
bucol, both compounds inhibited LDL oxidation and both reduced atherosclerosis
consistent with an antioxidant-mediated reduction in lesion formation. Subsequent
studies in primates (Sasahara et al., 1994), cholesterol-fed rabbits (Shaish et al.,
1995), and hamsters (Parker et al., 1995) have also demonstrated a reduction in
atherosclerosis with probucol.
Other antioxidants have also been tested for their ability to inhibit atherosclerosis.
Once such compound is N ; N 0 -diphenyl-phenylenediamine (DPPD), an aniline
compound that inhibits LDL oxidation ex vivo. Inclusion of DPPD in the diet of
cholesterol-fed rabbits signicantly reduces atherosclerosis and cholesterol accu-
mulation in the aorta (Sparrow et al., 1992). This nding is also associated with
enhanced LDL resistance to oxidation ex vivo (Sparrow et al., 1992). Similar ndings
have been extended to a murine model of atherosclerosis with a signicant inhibition
of atherosclerosis and LDL oxidation in apo E (/) mice fed a high-fat diet
(Tangirala et al., 1995). Similarly, combined supplementation of butylated hy-
droxytoluene and butylated hydroxyanisole (two lipid-soluble antioxidant com-
pounds) prevents atherosclerosis in butter-fed rabbits (Wilson et al., 1978), whereas
butylated hydroxytoluene alone reduces atherosclerotic lesions in cholesterol-fed
rabbits (Byorkhem et al., 1991). Finally, Cynshi and co-workers (Cynshi et al., 1998)
have tested a synthetic compound (BO-653) that contains structural components of
both vitamin E and probucol for its eect on atherosclerosis. In both rabbit and
murine models of atherosclerosis, BO-653 inhibits atherosclerosis signicantly
(Cynshi et al., 1998).
Thus, a number of lipid-soluble antioxidant compounds have been used to
demonstrate an association between ex vivo, inhibition of LDL oxidation, and a
reduction in atherosclerosis. Not all studies have been positive, and a number of
inferences between antioxidant protection and inhibition of atherosclerosis have
been indirect. This lack of direct evidence has presented a problem for the oxidative
modication hypothesis (for review, see Stocker, 1999).
2.4.7. Problems with the oxidative modication hypothesis

Despite the evidence outlined above supporting the role of LDL oxidation in
atherosclerosis, some skepticism has arisen concerning an absolute requirement for
LDL oxidation to initiate atherosclerosis. In particular, there has been diculty
consistently linking the resistance of LDL to oxidation with the antiatherogenic
action of certain antioxidants. For example, although probucol inhibits both LDL
oxidation and atherosclerosis in cholesterol-fed (Shaish et al., 1995; Daugherty
et al., 1989), and WHHL (Kita et al., 1987; Carew et al., 1987; Nagano et al.,
1992) rabbits, vitamin E provides signicant protection against LDL resistance to
oxidation without any eect of atherogenesis in the very same models (Shaish et
al., 1995; Fruebis et al., 1995; Kleinveld et al., 1995). These ndings imply that
manipulating ex vivo LDL resistance to oxidation does not necessarily translate
into reduced atherosclerosis. Another complicating matter is the observation in
one study demonstrated a reduction of atherosclerosis with b-carotene in choles-
terol-fed rabbits without any change in LDL resistance to oxidation (Shaish et al.,
1995). Thus, there is no secure evidence that measurements of ex vivo LDL re-
sistance to oxidation predict the antiatherogenic activity of lipid-soluble antioxi-
dants.
One major problem with this analyses is linking an ex vivo assay (LDL resistance
to oxidation) to events that occur within the vascular wall. To address this question,
Witting and colleagues examined lipid peroxidation within atherosclerotic aorta and
tested the eect of both probucol and its metabolite, bisphenol on atherosclerosis
and lipid peroxidation in WHHL rabbits (Witting et al., 1999). In this study, both
bisphenol and probucol enhanced the resistance of circulating LDL to oxidation.
Both compounds strongly inhibited aortic accumulation of lipid oxidation products,
however, only probucol had an inhibitory eect on atherosclerosis whereas bisphenol
did not. More importantly, the extent of atherosclerosis in this study did not cor-
relate with the aortic content of oxidized lipids. This study suggests that aortic ac-
cumulation of oxidized lipids is not a prerequisite for the initiation and progression
of atherosclerosis in WHHL rabbits. These data question the absolute requirement
for LDL oxidation in the initiation of atherosclerosis, and suggest a further study is
needed to precisely dene of oxLDL in atherosclerosis.
2.4.8. Clinical studies

A number of clinical trials have been conducted to explore the relation between
antioxidant status and atherosclerosis and selected examples are contained in Table 6.
There is currently a wealth of epidemiological data examining the interaction
between antioxidant intake, or antioxidant status in the development of vascular
disease. The data consists of a collection of descriptive studies, case-controlled
studies, prospective cohort studies, and a limited, but growing number of random-
ized trials.
2.4.9. Evidence from descriptive studies

Descriptive studies examine the characteristics of a population and its associated
disease rates, and compare the data from one time period, or one country to the next.
In general these studies suer from the inability to control for potentially con-
founding factors such as dierences in diet, genetics, and environmental factors.
However, these studies may be quite valuable as they can generate hypotheses to be
tested by more rigorous epidemiological means.
A total of ve descriptive studies published since 1975 have shown in inverse
association between fresh fruit and vegetable consumption in cardiovascular disease
Table 6
Selected studies and randomized trials of antioxidants and vascular disease
Study Study population Findings
Descriptive studies
Verlangieri et al. (1985) United States Inverse association of fruit and
vegetable consumption and
cardiovascular mortality rate
Gey and Puska (1989) Sixteen European regions Inverse association of mean plasma
vitamin E concentrations and
cardiovascular mortality
Case-control studies
Riemersma et al. (1989) 110 cases of angina and 394 Lower plasma vitamin E
normal subjects concentrations in cases vs. controls
Ramirez and Flowers 101 cases of CAD on angiogram Lower leukocyte ascorbic acid levels in
(1980) and 49 normal subjects cases vs. controls
Prospective studies
Nurses' Health Study 87,000 US female nurses Inverse association between CAD
(Stampfer et al., 1993) events and intake of vitamin E
Health Professionals' 39,000 US male Inverse association between CAD
Follow-up Study (Rimm Health professionals events and vitamin E or b-carotene
et al., 1993) intake*
NHANES Study 11,349 US men and women Inverse association between
(Enstrom et al., 1992) cardiovascular mortality and vitamin
C intake
Losonczy et al. (1996) 11,178 elderly US citizens Reduced CAD events in subjects
taking vitamin E vs. those not
taking vitamin E
Randomized, double-blind, Placebo-controlled trials
ATBC (The 2900 Finnish male smokers No eect of either b-carotene or
ATBCILCPSG, 1994) vitamin E on coronary artery disease
events
Physicians' Health Study 22,071 US male physicians No eect of b-carotene on CAD
(Hennekens et al., 1996) events
CHAOS (Stephens et al., 2002 British men and women 77% reduction in CAD events with
1996) with angiographic CAD vitamin E as compared with placebo
GISSI (GISSI 11,324 Patients with MI No eect of vitamin E, positive eect
Investigators, 1999) for PUFA
HOPE 9541 Patients at high risk No eect of vitamin E, positive eect
(Yusuf et al., 2000) for MI for angiotensin converting enzyme
inhibition with ramipril
rates (Acheson and Williams, 1983; Armstrong et al., 1975; Ginter, 1979; Gey and
Puska, 1989). In some cases these studies examined the intake of certain vitamins
(i.e., vitamin C, vitamin E) in cardiovascular disease mortality rates. The strongest
theme from these studies is an observed trend across populations that fresh fruit and
vegetable consumption tends to protect against cardiovascular disease. Whether this
is due to the dietary intake of antioxidants or the replacement by fresh fruits and
vegetables of potentially harmful dietary components (e.g., animal fats) cannot be
determined of (Gaziano, 1999).
2.4.9.1. Evidence from case control studies. Case control studies consist of data
gathered retrospectively on dietary or lifestyle exposures of interest as well as data on
a variety of potentially confounding variables. Individual cases are compared with
appropriate controls in an attempt to isolate the eect of a variable of interest from
potential confounders. Three such studies, one study each to support an association
between vitamin E (Riemersma et al., 1991), vitamin C (Ramirez and Flowers, 1980),
and tissue b-carotene levels (Kardinaal et al., 1993) in cases with cardiovascular
disease compared to controls, suggest that natural antioxidants reduce the risk of
cardiovascular disease. The strength of these conclusions is still limited, for example,
the selection of cases and controls may introduce bias, and the chosen controls may
also not adequately represent the intended population. Furthermore, uncharacter-
ized or unknown variables may have a signicant impact on results that go unex-
amined.
2.4.9.2. Evidence from prospective cohort studies. Prospective studies oer an ad-
vantage of measuring exposure of variables prior to the development of disease,
which minimizes the impact of selection and recall bias. This study design also
minimizes the eects that the disease may have on exposure related variables such as
dietary habits. Data from two such studies, the Nurses Health Study (Stampfer et al.,
1993), and the Health Professionals Follow-Up Study (Rimm et al., 1993) support an
inverse association between the development of coronary artery disease events, and
the intake of vitamin E. A third study conducted in elderly US citizens suggests an
inverse relation between coronary artery disease events such as heart attack and
stroke, and the dietary intake of vitamin E (Losonczy et al., 1996). Only one of these
studies, the Health Professionals Follow-Up Study (Rimm et al., 1993) supported
such an association for b-carotene. An association for other vitamins was not sup-
ported by these studies.
2.4.9.3. Randomized trials. A number of randomized trials have now been completed
which examine the eect of antioxidant supplementation on cardiovascular events.
Although not designed to look at cardiovascular events, the a-tocopherol, b-caro-
tene (ATBC) trial examined the eect of b-carotene in vitamin E on 29,000 nish
male smokers (The ATBCILCPSG, 1994). They found no eect of either vitamin on
coronary artery disease events although there was a pro-carcinogenic eect of b-
carotene in these smokers. Among the more celebrated antioxidant trials, the
Cambridge Heart Antioxidant Study (CHAOS) trial (Stephens et al., 1996) examined
the eect of vitamin E as secondary prevention for cardiovascular disease in 2002
British men and women with established coronary artery disease. In this trial
treatment with vitamin E was associated with a 77% reduction in non-fatal heart
attacks, but no change in mortality. Perhaps the most complete trial is the Heart
Outcomes Prevention Evaluation (HOPE) trial (Yusuf et al., 2000). In this trial over
9000 men and women were randomized to vitamin E or placebo and followed for a
mean of 412 years. In this study, there was no signicant eect of vitamin E to evaluate
the eects of cardiovascular disease at a vitamin E dose of 400 IU per day.
In summary then, there is no clear consensus that antioxidant supplementation

appears to ameliorate the eects of atherosclerosis. The precise reasons for these ob-
servations are not yet clear, although several possibilities exist. First, it may be true that
there is absolutely no relation between lipid peroxidation and the process of athero-
sclerosis. This seems highly unlikely given the available scientic evidence, however, it
may be true that the relative importance of lipid peroxidation is modest. Another
possibility relates to the dose response between antioxidants and any eect on car-
diovascular disease. It could be that in well-nourished populations most individuals
are already receiving the maximum benet of antioxidant treatment through a healthy
diet. A nal possibility is that only certain individuals are in a state of heightened
oxidative stress and would benet from antioxidant therapy. Since we have no good
assays for identifying those patients suering from excessive oxidative stress, it stands
to reason that indiscriminately treating patients with antioxidant therapy may not
facilitate the detection of an eect. For whatever the reason, it is clear that general,
routine, supplementation of individuals with lipid-soluble antioxidants in the hopes
that cardiovascular disease will be prevented does not appear to be a viable strategy.
2.5. The response-to-retention hypothesis of early atherogenesis
We have already discussed the problems associated with both the ``response-to-
injury'' and `òxidative modication'' hypotheses of atherosclerosis. A number of
studies suggest that retention of lipoproteins within the arterial wall has important
implications for atherogenesis. In general, the rate of LDL entry into normal seg-
ments of the arterial wall exceeds the rate of accumulation (Carew et al., 1984)
suggesting that LDL egress is the limiting factor for the accumulation of LDL in the
arterial wall. Moreover, the accumulation of atherogenic lipoproteins in the arterial
wall appears to be concentrated in areas predisposed to future lesion development
even though the rate of entry is similar to normal sites (Schwenke and Zilversmit,
1989a,b). These studies and several others (Schwenke and Carew, 1989a,b; Falcone
et al., 1984) indicate that lesion-prone arterial sites show an enhanced retention of
atherogenic lipoproteins containing apo B. These data suggest that it is the retention
of lipoproteins within the arterial wall that is the inciting event for atherosclerosis as
opposed to any oxidative modication of such lipoproteins.
2.5.1. Evidence to support the response-to-retention hypothesis

The central tenant of the response-to-retention hypothesis is that high plasma
concentrations of atherogenic lipoproteins are required and that persistent retention
within the arterial wall sets the stage for lesion development. Within two hours of
injecting LDL into rabbits, arterial retention of LDL and its micro aggregates can be
observed (Nievelstein et al., 1991). The precise mechanisms involved in the retention
of atherogenic lipoproteins within lesion-prone arterial sites is not clear. There is,
however, considerable evidence that components of the extracellular matrix partic-
ipate in this process. Apo B retained within the arterial wall is closely associated with
arterial proteoglycans (Yla-Herttuala et al., 1987; Camejo et al., 1993). Lipolytic
enzymes within the extracellular matrix also appear to play a role. For example,
lipoprotein lipase (LpL) enhances the adherence of LDL in vitro to proteoglycans

(Williams et al., 1991). This phenomenon appears to occur in vessels enriched with
LpL in vitro (Rutledge and Goldberg, 1994), and is independent of LpL enzymatic
activity (Williams et al., 1992a). Once retained within the arterial wall, LDL can
form micro aggregates perhaps through the action of secretory sphingomyelinase,
a normal component of the arterial wall (Xu and Tabas, 1991).
There are a number of important issues that arise from LDL that is entrapped within
the arterial wall. LDL bound proteoglycans appears to be more susceptible to oxida-
tion (Hurt-Camejo et al., 1992), and once oxidized, the LDL can obviously take on all
the proatherogenic activities discussed above for both ox-LDL. The retained LDL is
also subject to action by arterial wall sphingomyelinase (Xu and Tabas, 1991) that can
generate ceramides with a number of well documented eects such as induction of
apoptosis and mitogenesis (Hannun, 1994; Joseph et al., 1994). Most importantly,
aggregated LDL is avidly taken up by macrophages and smooth muscle cells (Ismail
et al., 1994) and thus, can support foam cell formation (Vijayagopal et al., 1992).
The precise nature of this aggregated LDL uptake by macrophages and smooth
muscle cells appears to involve receptors distinct from the LDL receptor (Vijayag-
opal et al., 1993). Interestingly, macrophage conversion to foam cells induces further
release of LpL (O'Brien et al., 1992) providing for enhanced retention of LDL and
further expansion of LDL aggregates within the arterial wall. A recent study lends
considerable credence to the response-to-retention hypothesis. Boren and colleagues
constructed a transgenic mouse strain overexpressing human apo B-100 and dem-
onstrated that dietary induction of hypercholesterolemia caused severe atheroscle-
rosis. In contrast, mice expressing a mutated form of apo B that does not bind
proteoglycans (but does bind the LDL receptor) develop almost no lesions despite a
similar degree of hypercholesterolemia (Boren et al., 1998). These results tend to
suggest that LDL association with proteoglycans is an important initial step in the
formation of atherosclerotic lesions.
3. The concept of disease activity
Thus far we have reviewed a number of theories involved in the initiation events
of atherosclerosis. Although dierent in some respects, all of these theories involve
the eventual recruitment of inammatory cells to the arterial wall and a response to
foam cell formation within the arterial intima. We have also reviewed briey the
events leading to lesion formation and the establishment of mature atherosclerotic
lesion. The ``classical'' mature atherosclerotic lesion (Fig. 3A) involves a central core
of foam cells with extracellular cholesterol arranged in so-called cholesterol ``clefts''.
There is typically also a considerable amount of necrotic debris, and overlying the
central core is a brous cap comprised of extracellular matrix, smooth muscle cells,
and collagen. Lesions may remain quiescent in this state for many years. Such lesions
may never produce any symptoms during the course of a patient's lifetime. At some
point, in order to eect atherosclerotic disease activity (i.e., heart attack or stroke),
the lesion must become activated. Lesion activation is initiated by rupture of the
Fig. 3. Morphological features of atherosclerotic lesions. (A) The ``classical'' atherosclerotic lesions
consists of a lipid core, with a brous plaque. The core often contains cholesterol crystals and necrotic
debris. Stable atherosclerotic plaques contain cells and extracellular matrix in a thick brous cap. In
contrast, (B) unstable plaques contain fewer cells and matrix in a thinner cap with an abundance of
macrophages in the ``shoulder'' regions of the cap.
atherosclerotic plaque such that the plaque contents are exposed to the luminal
surface of the artery (Fig. 3B). Contact with blood components in the plaque then
leads to a thrombotic response that may precipitate a vascular event. In the fol-
lowing section we will review what is known about lesion activation and the events
that contribute to this process.
3.1. The brous cap
For many years, end-organ ischemia was thought to result as a consequence of

progressive narrowing of the arterial lumen and loss of blood ow. Although rupture
of the atherosclerotic plaque was proposed in the rst half of this century (Leary,
1934), it is only in the past 15 yrs that this phenomenon has been appreciated in the
genesis of clinical events in atherosclerosis. Autopsy studies have consistently iden-
tied a number of morphologic features that are associated with recently ruptured
atherosclerotic plaque. These include a large necrotic core of lipid and cellular de-
bris, a thin brous cap that is often eccentric, and often a relatively modest pro-
trusion into the arterial lumen (Davies and Thomas, 1985; Davies et al., 1989). One
theory for these morphological characteristics concerns mechanical stresses on the
brous cap. In particular, the presence of a large, soft lipid core compromises the
structural integrity of the cap as the core is unable to bear mechanical forces and
focuses available forces on the brous cap (Richardson et al., 1989). A large portion
of these forces are concentrated at the junction of the brous cap with a normal
vessel, the so-called ``shoulder'' region (Fig. 3A and B). This region of the plaque,
coincidentally, is the region most likely to rupture based on autopsy studies (Davies
and Thomas, 1985). Although these are common features of atherosclerotic plaques
that have ruptured, not all plaques with this morphology will indeed fracture. In fact,
only small proportion of atherosclerotic plaques are vulnerable to rupture, and this
fact underlies the diculty in developing an adequate animal model for this phe-
nomenon. Nevertheless, the vascular biology of the brous cap has come into focus
in recent years, and has provided insights into modulating atherosclerotic lesions,
thus, making them more stable and less prone to rupture.
Among the major components of the atherosclerotic plaque is extracellular ma-
trix. Normally, extracellular matrix production and turnover is remarkably slow
(Seyer and Kang, 1992). In atherosclerosis, however, the environment of injury is
associated with enhanced synthetic activity of major matrix components such as
elastin, collagen, and proteoglycans (Libby, 1995). The population of inammatory
cells such as foam cells, and monocyte-derived macrophages within the plaque
produces an environment that is replete with the cytokines and growth factors. A
number of these agents have important implications for matrix production as some
cytokines, such as transforming growth factor-b (TGF-b) may stimulate collagen
synthesis whereas others (interferon-c) suppress it (Folcik et al., 1995). One proposed
mechanism of cap weakening is an insucient production of matrix within the cap.
3.2. Matrix degradation
Once a brous cap is formed over an atherosclerotic lesion, it is subject to a

number of stresses due to both blood pressure and shear on the luminal surface of
the artery. In the best of circumstances this cap can withstand such forces based
upon its structural integrity. A wealth information now indicates that the structural
components of the brous cap be degraded by a variety of matrix-degrading en-
zymes. These enzymes include serine proteases tissue-type and urokinase-type
plasminogen activators, plasmin, the matrix metalloproteninases (MMPs), and
cysteine proteases. Tables 7 and 8 contains a number of known matrix-degrading
enzymes, their locations and typical substrates. Since the matrix metalloproteninases
are capable of degrading all of the major components of the extracellular matrix,
Table 7
Matrix-degrading enzymes relevant to atherosclerosisa
Type Examples Examples of substrates
Serine protease Plasmin, urokinase, Fibrin, bronectin, laminin, some
cathepsin G, TPAb proteoglycans
Cysteine protease Cathepsins, B, D, H, L, N, Wide range, including collagen,
and S proteglycans, and elastin
Matrix Interstitial collagenase Collagens I, II, III, VII, and X
metalloproteinases (MMP-1)
Gelatinase A (MMP-2) Collagens IV, V, VII, and X
Stromelysin-1 (MMP-3) Collagens III, IV, V, and IX; laminin,
bronectin, elastin, proteoglycans
PUMP-1 (MMP-7) Gelatin, bronectin, laminin, collagen
type IV, procollagenase, and proteoglycan
core protein
Neutrophil collagenase (MMP-8) Collagens I, II, and III and proteoglycans
Gelatinase B (MMP-9) Collagens IV, V, VII, and X
Stromelysin-2 (MMP-10) Collagens III, IV, V, and IX; laminin,
bronectin, elastin, proteoglycans
Stromelysin-3 (MMP-11) Gelatin, bronectin, and proteoglycans
Metalloelastase (MMP-12) Elastin
MT-MMP (MMP-14) Collagen IV, gelatin, and progelatinase A
a
Adapted from Libby and Lee (1997).
b
Tissue-type plasminogen activator.
Table 8
Human studies of cholesterol lowering therapy and endothelial vasomotor functiona
Study rst author Baseline total Intervention Treatment Improvement
cholesterol (mg/dl) duration (mo) demonstrated
Coronary circulation
Leung et al. (1994) 275 31 Cholestyramine 6 +
Egashira et al. (1994) 272 8 Pravastatin 6 +
Treasure et al. (1995) 230 33 Lovastatin 5.5 +
Anderson et al. (1995) 209 33 Lovastatin plus 12
cholestyramine
Yeung et al. (1996) 204 32 Simvastatin 6 )
Forearm circulation
O'Driscoll et al. (1997) 255 33 Simvastatin 1 +
Drury et al. (1996) 209 17 Pravastatin 5 yrs +
Vogel et al. (1995) 200 12 Simvastatin 3 +
Tamai et al. (1997) 195 34 LDL apheresis Immediate +
a
Adapted from Dillon and Vita (2000).
there under a multiple levels of regulation. All are under transcriptional regulation
that has not been particularly well characterized. After synthesis, however, the
MMPs typically must be activated through cleavage of the N-terminus by the
activity of serine proteases such as plasmin or urokinase (Werb et al., 1977). A nal
level of regulation involves a class of proteins known as the tissue inhibitors of
metalloproteninases (TIMPs) (DeClerk et al., 1994). These proteins bind to matrix

metalloproteninase active sites and inhibit the enzymes.
With respect to the integrity of the brous cap, a number of matrix metallo-
proteninases have been observed in atherosclerotic plaques. For example, human
lesions contain MMP-3 and MMP-1 (Henney et al., 1991), with the former
demonstrating intense staining in the shoulder regions of atherosclerotic plaques
(Nikkari et al., 1995). The synthesis of MMPs within the vascular wall is now an
area of intense investigation. Smooth muscle cells and culture predominantly ex-
press MMP-2, a gelatinase that participates in cellular migration and only degrades
non-brillar collagen (Galis et al., 1994). Under resting conditions, cultured smooth
muscle cells express little of the enzymatic activity required to cleave brillar col-
lagen (MMP-1). One must keep in mind, however, that the atherosclerotic plaque is
typically bathed in a milieu of cytokines and growth factors. Under these condi-
tions, smooth muscle cell production of MMPs is quite dierent. In the presence of
tumor necrosis factor-a or interleukin-1b, vascular smooth muscle cells readily
produce MMP-1, and MMP-3, two enzymes capable of degrading structural col-
lagen (Table 7). Other cell types within the atheroma have also been implicated in
MMP production. Macrophages from arterial lesions in rabbits demonstrate pro-
duction of MMP-1 and MMP-3, consistent with cell culture ndings (Galis et al.,
1995). This activity of macrophages may important implications for plaque
strength as there appears to be an inverse correlation between macrophage density
and the mechanical strength of human aortic plaques (Lendon et al., 1991).
Consistent with this notion, macrophage inltration of the brous cap and its
shoulder regions is a common nding in morphologically unstable lesions (van der
Wal et al., 1994; Moreno et al., 1992).
Rupture of the atherosclerotic plaque leads to the exposure of the lipid core to the
blood vessel lumen. This has implications for promoting disease activity in athero-
sclerosis as the lipid core has a number of important features. For example, the
matrix derived from the core of atheromatous lesions is considerably more
thrombogenic in vitro than matrix derived from other parts of the arterial wall
(Fernandez-Ortiz et al., 1994). One possible reason for this observation is the
abundant content of tissue factor within the core of atherosclerotic lesions (Libby,
1995). Tissue factor activates factors IX and X after binding to the coagulation
factor VIIa (Nemerson, 1995), thereby activating the clotting cascade.
In addition to matrix degradation, depopulation of the brous cap may also
contribute to plaque rupture (Falk, 1992). In particular, brous caps that demon-
strate rupture tend to have an imbalance between the number of macrophages and
smooth muscle cells with many more macrophages than stable plaques and many
fewer smooth muscle cells (Falk, 1992). One explanation for this observation is that
activated T lymphocytes are also found within atherosclerotic plaques (Hansson
et al., 1989). A major cytokine derived from T cells is interferon-c, and vascular
smooth muscle cells exposed to cytokines such as interferon-c demonstrate apoptosis
(Bennett et al., 1995; Geng et al., 1996). One could certainly envision a situation
where inammation in specic areas of the atherosclerotic plaque may promote
smooth muscle cell apoptosis, and confer some defect in the synthetic capacity of the
atherosclerotic plaque. This combined with shear forces on the plaque cap could
promote rupture in the shoulder regions.
If one assumes that plaque vulnerability is determined, in part, by the activity of
matrix-degrading enzymes, one would predict manipulations known to reduce to
cardiovascular events should also reduce matrix-degrading enzyme activity. This
prediction has proven to be true in experimental animals. Aikawa and colleagues
(Aikawa et al., 1998) produced experimental atherosclerosis in rabbits by balloon
injury and an atherogenic diet. Rabbits were then continued on diets with or without
high cholesterol. Animals continuing on the high cholesterol diet demonstrated le-
sions consisting of many macrophages, and considerable MMP-1 expression. In the
group that did not continue on high cholesterol diet, proteolytic activity decreased
substantially and the aortic content of interstitial collagen increased. These results
suggest that cholesterol-lowering prompts a change in the arterial wall characterized
by lower matrix metalloproteinases activity and increased collagen retention. These
features would be expected to provide plaque stabilization.
In summary, development of the atherosclerotic lesion involves the generation of
a brous cap that overlies a lipid core. In the best of circumstances, this brous cap
remains thick and replete with smooth muscle cells that produce collagen adding to
the stability of the plaque. Under these circumstances, shear forces and mechanical
stress from arterial pressure on the plaque are not met with any structural failure,
and the lipid core remains isolated from the circulation. However, under certain
circumstances often associated with inammation and cytokines, components of the
plaque may change such that the shoulder regions of the atherosclerotic plaque
become replete with macrophages and matrix metalloproteinases activity. In addi-
tion, T cells populating the plaque may produce cytokines that promote apoptosis of
vascular smooth muscle cells. Under this scenario, the plaque becomes more acel-
lular and contains less interstitial collagen. This sets the stage for structural failure of
the plaque during the periods of high mechanical stress leading to plaque rupture
and exposure of the lipid core to the circulation. Since the lipid core contains a
number of prothrombotic components, this prompts thrombosis that may then
propagate to include the entire arterial lumen and produce vascular insuciency
with catastrophic consequences such as heart attack or stroke. Further work will be
needed to precisely identify the events of plaque instability and allow us to devise
strategies to promote integrity of the brous cap and prevent plaque rupture.
4. Vascular homeostasis
Although the section above makes a compelling case that we do not understand
all of the events associated with plaque rupture, it is clear that lesion activation also
involves a fundamental lapse in the homeostatic mechanism of the blood vessel
(Keaney and Vita, 1995; Levine et al., 1995). Normal vascular homeostasis provides
for adequate end-organ perfusion through the continuous control of vascular tone,
blood ow, and constitutive inhibition of thrombosis. The endothelium, as the
interface between blood ow and vessel wall, is an important and critical component
of vascular homeostasis. Among the more important features of the endothelium is

its role as a local site for the integration of paracrine and autocrine signals. For
example, the endothelium can respond to cytokine production within the vascular
wall by up regulating adhesion molecules on its surface that help promote leukocyte
recruitment into the blood vessel (Luscinskas and Gimbrone, 1996). The endothe-
lium also constitutively produces substances such as prostaglandin I2 PGI2 , a po-
tent inhibitor of platelet activation (Radomski et al., 1987). Endothelial cell synthesis
of heparins and thrombomodulin also promote blood uidity through the reduction
of thrombin activity at the endothelial surface (Ursini et al., 1995; Avissar et al.,
1989). Finally, the endothelium exerts control over brinolysis through the synthesis
of both tissue-type plasminogen activator, and plasminogen activator inhibitor-1
(PAI-1) (Loskuto and Edgington, 1977). Thus, the normal activity of the endo-
thelium maintains a state favoring brinolysis over thrombosis.
Another product of the vascular endothelium that maintains blood ow and
vascular tone is nitric oxide (NO), this species is a free radical synthesized constit-
utively in the endothelium by an oxido-reductase enzyme known as endothelial nitric
oxide synthase (eNOS). A number of important homeostatic functions appear to
depend on endothelium-derived NO. For example, vascular tone (Quyyumi et al.,
1995), smooth muscle cell phenotype (Garg and Hassid, 1989), platelet adhesion
(Azuma et al., 1986), and leukocyte recruitment (Kubes et al., 1994) all appear to be
controlled, at least in part, by nitric oxide from the endothelium. Experimental
animals lacking the eNOS exhibit spontaneous hypertension (Olesen et al., 1988), an
enhanced thrombotic response (Freedman et al., 1996), and defective vascular re-
modeling (Rudic et al., 1998). In patients, impaired nitric oxide action has been
associated with spontaneous arterial thrombosis (Freedman et al., 1996). Thus, a
predominance of available evidence indicates that the production of NO from the
endothelium is an important component of vascular homeostasis.
Nitric oxide elicits its bioactivity through two principal mechanisms. The rst
involves the binding of NO to the heme moiety of soluble guanylyl cyclase resulting
in enzymatic activation and the synthesis of 30 ,50 -cyclic guanosine monophosphate
(cGMP) (Arnold et al., 1977). There is an association between vascular cyclic GMP
and vasodilation due to nitric oxide (Ignarro et al., 1984) as well as NO-mediated
inhibition of platelet activity (Moro et al., 1995; Radomski et al., 1997). Another
mechanism of NO bioactivity relates to the formation of adducts of nitric oxide.
Under aerobic conditions NO forms oxides of nitrogen that combine with thiol
groups to form S-nitrosothiols (Wink et al., 1993), and this reaction has been im-
plicated in NO-mediated activation of potassium channels (Bolotina et al., 1994),
calcium channels (Xu et al., 1998), and the modulation of hemoglobin oxygen
anity (Stamler et al., 1997).
The component of vascular homeostasis contributed by nitric oxide has been the
subject of intense investigation with respect to atherosclerosis. The major reason
being that endothelium-derived NO bioactivity is impaired in patients who dem-
onstrate frank atherosclerosis, or known risk factors for premature atherosclerosis.
The vasodilatory responses to agents that stimulate endothelium-derived NO are
abnormal in the coronary arteries of patients with atherosclerosis (Ludmer et al.,
1986), diabetes (Nitenberg et al., 1993), and hypertension (Panza et al., 1993). This
appears to be a systemic process as NO-mediated responses are also impaired in
forearm resistance arterials (Panza et al., 1993; Creager et al., 1990; Johnstone et al.,
1992), as well as other conduit arteries such as the brachial artery (Anderson et al.,
1989; Celermajer et al., 1993). This abnormality in vascular homeostasis typically
precedes the clinical manifestations of vascular disease. Individuals with hypercho-
lesterolemia (Vita et al., 1990), advanced age (Celermajer et al., 1994), and cigarette
smoking (Kugiyama et al., 1996) typically demonstrate impaired vasoactive re-
sponses to agents that stimulate endothelial NO production. Genetic factors may
also play a role as patients with a family history have atherosclerosis also demon-
strate impaired NO-mediated arterial vasodilation (Clarkson et al., 1997). Thus,
atherosclerosis is characterized by a broad defect in vascular homeostasis that also
includes abnormal NO-mediated vascular homeostasis. This defect is thought to play
an important role in the clinical manifestations of vascular disease as patients with
abnormal vascular function are more likely to develop vascular events than those
whose vascular function is relatively well-preserved (Suwaidi et al., 2000; Schach-
inger et al., 2000).
4.1. Oxidative stress and impaired NO bioactivity
The exact mechanism producing abnormal bioactivity in the setting of vascular

disease and atherosclerosis remains a subject of intense investigation. However,
considerable evidence now indicates that NO bioactivity is particularly sensitive to
oxidative stress. Although oxidative stress is a poor term, it has come to gain ac-
ceptance as a means to describe an imbalance between oxidants and antioxidants in
favor of the former. With respect to vascular disease and atherosclerosis, there is
considerable evidence that there is an increase in the production of oxidants, and this
has important implications for NO bioactivity. The principal oxidants involved in
impairment of NO bioactivity will be discussed in detail below focusing on the
mechanistic aspects by which NO bioactivity is impaired.
4.1.1. Superoxide and NO bioactivity

It has been known for some time that atherosclerotic blood vessels demonstrate
abnormal functional responses (Henry and Yokoyama, 1980; Heistad et al., 1984)
and the knowledge that endothelium-dependent vasorelaxation is due, in part, to
nitric oxide (Ignarro et al., 1987) has fueled considerable investigation to determine
the mechanisms responsible for impaired endothelial function in atherosclerosis. To
gain insight into the mechanism of vascular dysfunction and atherosclerosis, Minor
and colleagues (Minor et al., 1990) measured the elaboration of nitrogen oxides from
normal, hypercholesterolemic, and atherosclerotic blood vessels. They then corre-
lated this with the amount of relaxation produced by those blood vessels and the
result is shown is Fig. 4. Diseased blood vessels exhibit considerably less relaxation
per unit of nitrogen oxide released suggesting that NO may be inactivated in
pathologic states. This source of NO inactivation has since been identied as su-
peroxide (Ohara et al., 1993; Keaney et al., 1995), and a number of disease states
Fig. 4. Arterial relaxation and NO production in rabbit aorta. Rabbits were treated with a 1% cholesterol
diet for either 25 weeks (hypercholesterolemia) or 4 months (atherosclerotic). Arterial relaxation and
NOx (nitrite+nitrate) were examined in response to 1 lM acetylcholine or 10 lM calcium ionophore
A23187. Reproduced from The Journal of Clinical Investigation (1990) 86:21092116 by copyright per-
mission of the American Society for Clinical Investigation.
such as hypercholesterolemia, diabetes, and hypertension are now noted to be as-

sociated with an increase in the steady state ux of superoxide within the vascular
wall (Griendling and Alexander, 1997). This change in the ambient superoxide level
has important implications for nitric oxide. It is known that superoxide and NO
combine rapidly in a radical-radical combination reaction that occurs at the diusion
limit k 1:9 1010 M1 s1 (Kissner et al., 1997) to form peroxynitrite as depicted
in reaction (9)
O
2 NO ) OON O

9
The relevance of this reaction on NO bioactivity is emphasized by experimental

evidence that it occurs in vivo (Leeuwenburgh et al., 1998; Ischiropoulos et al., 1992).
The product of this reaction, peroxynitrite, has the capacity to activate guanylyl
cyclase (Mayer et al., 1995; Tarpey et al., 1995), although it does so much less po-
tently than nitric oxide itself (Tarpey et al., 1995). Therefore, any interaction of NO
and superoxide in the vascular wall will result in a reduction of NO bioactivity.
Since endothelial cells produce NO and superoxide, small changes in the relative
ux of either species should have important implications for NO bioactivity. This
prediction has been proven true in experimental systems proving that SOD improves
the vascular relaxation response to endothelium-derived NO (Rubanyi et al., 1986;
Gryglewski et al., 1986). Both receptor-stimulated NO release from endothelial cells
as well as the basal constitutive production appear to be responsive to ambient levels
of superoxide (Rubanyi et al., 1986; Gryglewski et al., 1986). Further evidence that
ambient superoxide is important for NO bioactivity comes from experiments in
which endogenous SOD activity has been inhibited. For example, inactivation of
CuZn SOD through chelation with diethyldithiocarbamate results in impaired

release of bioactive NO (Omar et al., 1991). Chronic inhibition of CuZn SOD
through copper-deciency also results in impaired NO-mediated arterial relaxation
(Lynch et al., 1997; Schuschke et al., 1992) that is associated with an increased
vascular ux of superoxide detected by chemiluminescence (Lynch et al., 1997).
Available evidence, therefore, indicates that vascular SOD activity is an important
component for intact NO responses.
Despite the consensus that ambient superoxide helps determine NO bioactivity,
considerable controversy exists as to the source of superoxide especially in the setting
of vascular disease. All layers of the vascular wall have demonstrated evidence for
superoxide production (Pagano et al., 1995; Pagano et al., 1997; Miller et al., 1998).
Immunohistochemical data suggests that at least in the aortic adventitia, there is
evidence for NADPH oxidoreductase activity (Pagano et al., 1995) and the presence
of known subunits of the neutrophil NADPH oxidase such as p22phox , gp91phox ,
p47phox , p67phox (Pagano et al., 1997). Other studies in cultured endothelial cell
homogenates indicate that NADH oxidoreductase is a principal source of superoxide
(Mojazzab et al., 1994).
In the setting of vascular disease, other sources of superoxide have been impli-
cated including xanthine oxidase (Ohara et al., 1993) that may actually be bound to
the surface of the endothelium (White et al., 1996). This nding would be consistent
with studies in patients with hypercholesterolemia that demonstrate a partial im-
provement in NO-mediated arterial relaxation with an inhibitor of xanthine oxidase,
oxypurinol (Cardillo et al., 1997). The signaling events involved in stimulating
NADPH oxidase activity in atherosclerosis are now being elucidated. Warnholtz and
colleagues found that superoxide production in cholesterol-fed rabbits was increased
signicantly, and this response could be inhibited by an angiotensin-II type I re-
ceptor antagonist (Warnholtz et al., 1999). These results suggest that angiotensin-II,
at least in part, mediates the increase in superoxide production observed with
hypercholesterolemia.
In considering the sources of superoxide, one must consider that nitric oxide
synthase is capable of reducing molecular oxygen to produce superoxide (Pou et al.,
1992; Vasquez-Vivar et al., 1998; Xia et al., 1998a). This may have particular im-
portance for hypercholesterolemia-induced vascular superoxide generation as en-
dothelial cells exposed to LDL exhibit enhanced superoxide generation from eNOS
(Pritchard et al., 1995). The precise mechanisms underlying these observations are
not yet clear, but may have to do with depletion of intracellular tetrahydrobiopterin
BH4 , a co-factor for eNOS that appears important in determining relative super-
oxide production from this enzyme (Vasquez-Vivar et al., 1998).
Although considerable data suggests that vascular superoxide is an important
determinant of NO bioactivity, it is clear that the entire spectrum of impaired NO
bioactivity in hypercholesterolemia and atherosclerosis cannot be attributed solely to
superoxide. For example, even in studies reporting improved NO bioactivity by
increasing vascular superoxide dismutase (Mugge et al., 1991; White et al., 1994), the
eect on NO bioactivity was incomplete. Consistent with these observation, chronic
SOD inhibition appears to impair receptor-dependent stimuli for NO production to
a greater extent than non-receptor-dependent stimuli (Lynch et al., 1997). This

nding suggests that NO elaboration may also be impaired during later stages of
atherosclerosis (Oemar et al., 1989). One such candidate for impaired NO elabora-
tion is lipid peroxidation.
4.1.2. Lipid peroxidation and NO bioactivity

It is important to realize that other sources of oxidative stress are present in
atherosclerosis beyond superoxide. In particular, peroxynitrite is readily formed
from superoxide and NO and this product is a potent oxidant (Beckman and Crow,
1993). As an oxidant, peroxynitrite has a number of important activities including
the transfer of oxygen atoms (Beckman and Crow, 1993), oxidation of sulfhydryls
(Moreno and Pryor, 1992), and the initiation of lipid peroxidation (Graham et al.,
1993). Other important sources of oxidative stress beyond peroxynitrite may include
redox-active copper from ceruloplasmin (Ehrenwald et al., 1994), tyrosyl radical
from myeloperoxidase (Heinecke et al., 1993; Heinecke, 1997), lipoxygenase (Folcik
et al., 1995), and hypochlorous acid, another product of myeloperoxidase (Malle
et al., 1995).
The process of lipid peroxidation (Fig. 2) is of some consequence for NO bio-
activity. Lipid peroxyl radicals produced during this process combine readily with
peroxynitrite to form lipid peroxynitrite derivatives (Padmaja and Huie, 1993;
Rubbo et al., 1994) eectively quenching bioactive NO (O'Donnell et al., 1999).
Lipid peroxidation also leads to the formation of oxLDL (Yla-Herttuala et al., 1989)
that may either directly inactivate NO (Chin et al., 1992) or reduce eNOS in en-
dothelial cells (Liao et al., 1995). Indirect eects of lipid peroxidation include
interruption of G-protein-coupled signal transduction leading to impaired NO
production (Kugiyama et al., 1990). Thus, lipid peroxidation has a number of
important consequences for NO bioactivity.
4.1.3. Lipid-soluble antioxidants and NO bioactivity

In light of the aforementioned evidence that lipid peroxidation impairs NO bio-
activity, it follows that antioxidants that reduce vascular lipid peroxidation should
improve NO bioactivity. As shown in Table 1, the main lipid-soluble antioxidants
are a-tocopherol (vitamin E), b-carotene, and ubiquinol-10. These three compounds
represent the main lipid soluble antioxidants in humans and, with the exception of
b-carotene (Jialal et al., 1991; Gaziano et al., 1995), their main action is thought to
be the inhibition of lipid peroxidation (Esterbauer et al., 1989).
4.1.3.1. a-Tocopherol and b-Carotene. The activity of a-tocopherol and b-carotene

with respect to NO bioactivity has been examined in rabbits consuming a 1% cho-
lesterol diet that typically demonstrate impaired endothelium-derived NO bioactivity
as assessed by arterial relaxation (Jayakody et al., 1985; Verbeuren et al., 1986). This
defect induced by cholesterol-feeding has been linked to an increase in vascular
oxidative stress (Ohara et al., 1993; Keaney et al., 1995). We found that cholesterol-
fed rabbits treated with either b-carotene or a-tocopherol demonstrate near normal
NO-mediated arterial relaxation responses to both acetylcholine and A23187
(Keaney et al., 1993). At least with a-tocopherol, this dietary treatment was asso-
ciated with enhanced antioxidant protection as assessed by lipoprotein resistance to
copper-mediated oxidation (Keaney et al., 1993). Similar studies in the coronary
(Andersson et al., 1994) and carotid (Stewart-Lee et al., 1994) arteries of cholesterol-
fed rabbits have also demonstrated that a-tocopherol preserves NO-bioactivity. In
rats, a species not normally susceptible to atherosclerosis, cholesterol-feeding has
been associated with impaired endothelium-dependent arterial relaxation and this
eect of cholesterol is abrogated by dietary vitamin E (Lutz et al., 1995).
The role of a-tocopherol in preserving NO bioactivity has not been limited solely
to atherosclerosis and hypercholesterolemia. For example, aortic segments isolated
from streptozotocin-induced diabetic rats demonstrate impaired NO-mediated ar-
terial relaxation that improves signicantly with a-tocopherol supplementation in
the diet (Keegan et al., 1995; Karasu et al., 1997a,b). Similar observations have been
reported in isolated perfused rat coronary arteries (Rosen et al., 1996). To the extent
that diabetes represents a state of heightened oxidative stress (Gopaul et al., 1995;
Davi et al., 1999; Keaney and Loscalzo, 1999), these data provide additional evi-
dence that antioxidant protection in vivo is important in maintaining normal NO
bioactivity in the setting of chronic vascular disease.
Since LDL oxidation impairs NO bioactivity (Kugiyama et al., 1990), and a-
tocopherol inhibits LDL oxidation (Dieber-Rotherneder et al., 1991), one might
speculate that a-tocopherol preserves NO bioactivity by inhibiting LDL oxidation.
Available evidence suggests that this contention may be overly simplistic. For ex-
ample, cholesterol-fed rabbits treated with two dierent regimens of a-tocopherol
demonstrates strikingly dierent eects on NO bioactivity. At an a-tocopherol dose
of 110 IU/d, NO-mediated arterial relaxation is preserved in cholesterol-fed rabbits
and LDL is protected from ex vivo copper-mediated oxidation (Keaney et al., 1993,
1994). In contrast, a 10-fold higher dose of a-tocopherol produces worse NO bio-
activity than cholesterol feeding alone despite excellent antioxidant protection of
LDL (Keaney et al., 1994). Thus, antioxidant protection of LDL alone is insucient
to preserve NO bioactivity in the cholesterol-fed rabbit model.
a-Tocopherol is a very ecient scavenger of lipid peroxyl radicals, nevertheless, it
is clear that atherosclerosis exhibits lipid peroxidation in the vascular wall despite the
presence of a-tocopherol (Suarna et al., 1995). In light of this knowledge, one fa-
vorable action of a-tocopherol may be the protection of vascular cells against the
deleterious eects of lipid peroxidation by-products. Consistent with this notion,
isolated arterial segments derived from a-tocopherol-decient rabbits demonstrate
impaired NO-mediated arterial relaxation upon exposure to ox-LDL (Keaney et al.,
1996). In contrast, arterial segments derived from animals supplemented with a-
tocopherol contain 100-fold more tocopherol and exhibit a marked resistance to the
eects of ox-LDL (Keaney et al., 1996). Similar eects can be demonstrated using
cultured endothelial cells (Jay et al., 1997). Recent data has shed light on the
mechanism of such observations. Exposure of arterial tissue to oxidized LDL results
in protein kinase C stimulation (Kugiyama et al., 1992) and this eect can be pre-
vented by incorporation of a-tocopherol into endothelial cells (Fig. 5) (Keaney et al.,
1996). Since protein kinase C stimulation impairs NO bioactivity (Sugiyama et al.,
Fig. 5. a-Tocopherol prevents oxLDL-induced protein kinase C stimulation in endothelial cells. Human
aortic endothelial cells were loaded with a-tocopherol (AT) and incubated with oxidized LDL (oxLDL),
native LDL (nLDL), or media (control) for 4 h. After washing, protein kinase C activity was determined
with an in situ assay (Williams and Schrier, 1992). Reproduced from The Journal of Clinical Investigation
(1996) 98:386394 by copyright permission of the American Society for Clinical Investigation.
1994; Ohgushi et al., 1993; Flavahan et al., 1991) and a-tocopherol prevents protein
kinase C stimulation (Keaney et al., 1996; Boscoboinik et al., 1991; Freedman et al.,
1996), these data point to the eect of a-tocopherol on protein kinase C as the
mechanism for improved NO bioactivity. It is worth noting that similar eects have
been observed in platelets (Freedman et al., 1996) and smooth muscle cells
(Boscoboinik et al., 1991).
4.1.3.2. Probucol. The eect of probucol to limit atherosclerosis has been discussed in
detail above. In the setting of atherosclerosis, supplementation with probucol results
in its accumulates within the vascular wall (Shaish et al., 1995; Keaney et al., 1995).
Since probucol accumulates in vascular tissue, and it is a potent inhibitor of LDL
oxidation in a variety of oxidizing systems (Marshall, 1982; Parthasarathy et al.,
1986), it is no surprise that its activity to modulate NO bioactivity has been exam-
ined.
Probucol has been tested for its eect on endothelium-derived NO in cholesterol-
fed (Keaney et al., 1995; Simon et al., 1993; Inoue et al., 1998) and LDL receptor-
decient (Hoshida et al., 1997) rabbits. With respect to its antioxidant activity,
probucol treatment as 1% of the diet did eliminate the increase in plasma (Simon
et al., 1993; Inoue et al., 1998) and aortic (Keaney et al., 1995) lipid peroxides asso-
ciated with cholesterol feeding as assessed by thiobarbituric acid-reactive substances.
In all the animal studies conducted thus far, probucol treatment universally pre-
served NO bioactivity measured as endothelium-dependent arterial relaxation in
response to either acetylcholine or A23187 (Inoue et al., 1998; Keaney et al., 1995;
Simon et al., 1993). In normal animals, probucol does not appear to alter NO
bioactivity (Simon et al., 1993), suggesting that its eect is limited to vascular disease
states. The eect of probucol is also not limited to atherosclerosis, since it also im-
proves NO bioactivity in alloxan-induced diabetes (Tesfamariam and Cohen, 1992).
Although probucol potently inhibits LDL oxidation in ex vivo assays (Marshall,
1982; Parthasarathy et al., 1986), its mechanism of action in these animal models
appears unrelated to this eect. Cholesterol-fed rabbits typically demonstrate an
increased vascular steady-state superoxide ux that can be detected by chemilumi-
nescence methods (Ohara et al., 1993). In two separate studies, probucol treatment
leads to a reduction in the increased vascular ux of superoxide (Keaney et al., 1995;
Inoue et al., 1998). This eect of probucol is not related to superoxide scavenging by
the compound (Keaney et al., 1995). Reducing the vascular ux of superoxide in-
hibits arterial wall lipid peroxidation (Keaney et al., 1995; Inoue et al., 1998) and
accumulation of lysophosphatidylcholine (Keaney et al., 1995), two phenomena that
have been linked to reduced NO bioactivity in animal models (Lynch et al., 1997;
Flavahan, 1992). Thus, probucol improves NO bioactivity in experimental animals
principally through a direct eect on arterial tissue. The precise nature of this eect is
not known, but may involve the signals needed to stimulate superoxide production.
Human studies examining the eect of lipid soluble antioxidants on NO bioac-
tivity are more mixed than animal studies. Studies examining antioxidant eects on
NO bioactivity in resistance vessels of patients with vascular disease (McDowell
et al., 1994; Elliott et al., 1995; Gilligan et al., 1994). In contrast, the eect of lipid-
soluble antioxidants on NO responses in most conduit arteries has been somewhat
more consistent. Koh and colleagues examined the eect of a-tocopherol on post-
menopausal women and observed an improvement in NO-mediated endothelium-
dependent vasodilation (Koh et al., 1998). Similar ndings have been reported in
patients with elevated remnant lipoprotein levels (Motoyama et al., 1998) in the
coronary circulation in response to vitamin E treatment. The reader is directed to a
recent review of human studies examining the eect of antioxidants on vascular
function (Duy et al., 1999).
4.1.4. Water-soluble antioxidants and NO bioactivity

With the popularity of the `òxidative modication hypothesis'', there has been
considerable investigation into water-soluble antioxidants and vascular function.
The principal water-soluble antioxidants in the arterial wall are glutathione (GSH)
and ascorbic acid. Glutathione is typically present in plasma at concentrations below
1 lM (Wendel et al., 1980), whereas ascorbic acid is normally considerably more
abundant (30150 lM) in plasma (Keaney and Frei, 1994). In contrast, both com-
pounds demonstrate relatively high concentrations within the cell cytosol reaching
concentrations that range from 1 to 5 mM (Bray and Taylor, 1993; Bergsten et al.,
1994). Considerable evidence suggests that both intracellular antioxidant com-
pounds are required for normal cellular viability and function. All cells appear to
demonstrate the active transport and/or synthesis of GSH and vitamin C and Ani-
mals deprived or depleted of either compound become morbid and may die as a
consequence (Martensson et al., 1993; Martensson and Meister, 1991).
4.1.4.1. Glutathione. The potential role of intracellular GSH in the bioactivity and
production of NO has been controversial. Early studies involving the manipulation
of intracellular GSH in porcine (Murphy et al., 1991) and bovine (Hecker et al.,
1992) endothelial cells produced concordant changes between GSH and the release
of NO, consistent with the role for GSH in endothelial cell NO production. How-
ever, specic correlation between the intracellular thiol levels and the release of NO
has been problematic. Nitric oxide production from cultured bovine aortic endo-
thelial cells by bradykinin is impaired by thiol alkylation with N-ethylmaleimide or
thiol oxidation with 2,20 -dithiodipyridine (DTDP). However, DTDP treatment was
not linked to a demonstrable reduction in endothelial cell GSH (Hecker et al., 1992),
suggesting some other activity of DTDP may interfere with NO release. In a separate
study with bovine cells, glutathione depletion using buthionine sulfoximine also
failed to demonstrate any reduction in bioactive NO despite a >90% reduction in
GSH (Mugge et al., 1991). Thus, these early studies in animals cells provided no
consensus that intracellular GSH is important in endothelium-derived NO produc-
tion.
Studies in human cells, however, have been more consistent with the role of in-
tracellular GSH in NO production. The treatment of human umbilical vein endo-
thelial cells with 1-chloro-2,4-dinitrobenzene (CDNB), a compound that covalently
modies GSH, reduced both intracellular GSH and endothelial NO production
(Ghigo et al., 1993). Conversely, increasing endothelial cell GSH with GSH ester
increased cellular NO production in a manner that strongly correlated with intra-
cellular GSH (Ghigo et al., 1993). It is dicult to reconcile the inconsistent ndings
of the in vitro studies outlined above. One possibility involves the non-specic action
of many thiol modulating agents. For example, although CDNB reduces intracel-
lular glutathione, it also has activity to inhibit thioredoxin reductase, an enzyme
important in maintaining protein thiols in a reduced state. Since protein thiols are
known to be important for eNOS activity (Patel et al., 1996), this may represent one
mechanism for observed ndings.
In contrast to the data obtained in vitro, emerging data from human studies in-
dicate that glutathione may have a role in modulating endothelium-derived NO.
Patients with documented atherosclerosis and coronary artery disease are charac-
terized by impaired NO-mediated arterial relaxation in the coronary (Ludmer et al.,
1986) and brachial circulations (Celermajer et al., 1992). Treatment of such patients
with L-to-oxo-4-thiazolidine carboxylate, an agent that selectively increases intra-
cellular glutathione (Williamson and Meister, 1981; Boesgaard et al., 1994), pro-
duced improved NO bioactivity in the brachial artery (Vita et al., 1998). These
ndings are consistent with recent evidence from resistance vessels in the femoral
circulation. Prasad and colleagues (Prasad et al., 1999) examined the eect of
glutathione in patients with atherosclerosis or its risk factors. Infusion of glutathione
markedly enhanced the response to aceylcholine, a receptor-mediated agonist for
NO production (Prasad et al., 1999). This enhancement of NO-mediated vasodila-
tion was associated with an increase in the plasma cGMP content of the femoral
vein, consistent with the notion that glutathione enhances NO bioactivity. Similar
results have been observed in the coronary circulation. For example, in patients with
a vasospastic angina, intracoronary glutathione improves NO bioactivity in response

to acetylcholine (Kugiyama et al., 1998).
Although emerging evidence supports a role for glutathione and NO bioactivity,
the mechanisms responsible for this eect remain to be elucidated. Early studies with
iNOS demonstrated that glutathione was necessary for optimal enzymatic activity
(Stuehr et al., 1990). More recent studies have conrmed this eect for neuronal
NOS (Komori et al., 1995; Hofmann and Schmidt, 1995). The eect of glutathione
appears unique as other thiols such as cysteine are considerably less eective (Ko-
mori et al., 1995; Hofmann and Schmidt, 1995). The precise events of this phe-
nomenon are best understood for neuronal NOS, where thiol-mediated enhancement
of enzymatic activity is associated with direct thiol binding to the heme moiety of
nNOS cooperatively with tetrahydrobiopterin (Gorren et al., 1997).
With respect to antioxidant activity of glutathione, one possible mechanism might
include the prevention of NOS inactivation by peroxynitrite that is produced during
simultaneously generation of NO and superoxide by the enzyme when substrate
conditions are limiting (Vasquez-Vivar et al., 1998; Fitzgerald et al., 1986). Recent
studies demonstrating that tissue GSH is reduced in the setting of hypercholester-
olemia resulting in enhanced oxidative damage tend to support this notion (Ma et al.,
1997).
4.1.4.2. Ascorbic acid. Ascorbic acid is a versatile antioxidant, it scavenges a wide

range of biologically relevant oxidizing species including superoxide, aqueous per-
oxyl radicals, hydrogen peroxide, and hydroxyl radical (Nishikimi, 1975; Bodannes
and Chan, 1979; Halliwell et al., 1987; Frei et al., 1989). As a consequence of this
potent antioxidant activity, the eect of ascorbic acid on NO bioactivity has been
examined. For example, coronary artery disease patients treated with oral ascorbic
acid demonstrate a signicant improvement in NO bioactivity within the brachial
artery and plasma concentrations of ascorbic acid remain within the normal range
(Levine et al., 1996). In contrast, a number of other studies have demonstrated a
salutary eect of ascorbic acid on NO-mediated arterial relaxation using pharma-
cological concentrations. For example, diabetic (Ting et al., 1996) or hypercholes-
terolemic (Ting et al., 1997) patients treated with high-dose intra-arterial ascorbic
acid (about 10 mM) demonstrated enhanced endothelial function in forearm resis-
tance vessels. Similar eects have been observed with other clinical conditions as-
sociated with atherosclerosis such as smoking (Heitzer et al., 1996), congestive heart
failure (Hornig et al., 1998), and hypertension (Koh et al., 1998; Motoyama et al.,
1998).
There has been considerable speculation concerning the mechanism of ascorbic
acid action. At rst glance, the fact that ascorbic acid scavenges superoxide suggest
that superoxide scavenging may be responsible for the improved NO bioactivity
(Koh et al., 1998; Ting et al., 1996; Ting et al., 1997; Heitzer et al., 1996; Hornig et al.,
1998). Direct studies in isolated arterial segments suggest a more careful inter-
pretation of the data is warranted. Isolated arteries exposed to superoxide demon-
strate impaired NO-mediated arterial relaxation that improves with physiologic
concentrations of SOD (about 13 lM) (Fig. 6) (Rubanyi et al., 1986; Jackson et al.,
Fig. 6. Ascorbic acid does not readily substitute for SOD in preserving NO bioactivity. (A) Contracted
vessels were assayed for NO bioactivity as relaxation in response to acetylcholine in the presence of no
additions (squares), superoxide from pyrogallol (circles), pyrogallol with 300 IU/ml SOD (diamonds), or
300 IU/ml SOD alone (down triangles). (B) NO bioactivity in response to acetylcholine in the presence of
no additions (squares), pyrogallol (circles), or pyrogallol with 0.1 (up triangles), 1.0 (diamonds), or 10
(down triangles) mM ascorbic acid. P < 0:05 vs no additions only by two-way ANOVA. Taken from
Jackson et al. (1998) with permission.
1998; Ignarro et al., 1988; O'Keefe et al., 1996). However, ascorbic acid cannot
substitute for SOD at physiologically relevant concentrations (Jackson et al., 1998).
The need for pharmacological concentrations of ascorbic acid can be predicted based
on available kinetic data considering that the interaction between superoxide and
ascorbic acid 105 M1 s1 occurs approximately 105 -fold less rapidly than the
interaction of superoxide with NO 1010 M1 s1 (Kissner et al., 1997). Thus,
simple superoxide scavenging appears to provide an incomplete explanation for the
eect of ascorbic acid on NO bioactivity.
The requirement for pharmacological concentrations of ascorbic acid in these
acute infusion studies has recently been demonstrated. Sherman and colleagues
(Sherman et al., 2000) observed that a high-dose of ascorbic acid (24 mg/min) in the
forearm improved NO-mediated arterial relaxation in hypertensive patients. In
contrast, a ten-fold lower dose designed to produce arterial concentrations of
ascorbate between 100 and 300 lM did not improve NO responses (Sherman et al.,
2000). These data suggest that for ascorbic acid to act through superoxide scav-
enging, pharmacological concentrations are needed in keeping with in vitro data
(Jackson et al., 1998).
The question of how ascorbic acid acts to improve NO bioactivity at normal
plasma concentrations (Levine et al., 1996) has recently been addressed. Incubation
of endothelial cells culture leads to the intracellular accumulation of ascorbate and
improved NO bioactivity (Heller et al., 1999; Huang et al., 2000). This eect appears
to be the result of increased synthesis of NO from endothelial cells as measured by
the accumulation of nitrogen oxides or enzymatic production of L -citrulline (Huang
et al., 2000). Interestingly, intracellular manipulation of glutathione status does not
have the same eect in this system. The reason for these observations stems from the
fact that increasing intracellular ascorbic acid also increases BH4 within the endo-
thelial cell, whereas glutathione manipulation has no eect in this regard (Huang
et al., 2000). Thus, intracellular ascorbic acid modulates endothelial cell NO
production through a BH4 -dependent mechanism.
4.1.5. Other means of treating endothelial dysfunction

4.1.5.1. L -arginine. After the initial description that endothelial dysfunction was a
prominent feature of patients with atherosclerosis (Ludmer et al., 1986), there was
considerable interest in identifying treatments that might reverse endothelial dys-
function in patients. Unlike those outlined above, such studies were not specically
directed at any particular hypothesis of atherosclerosis. For example, one of the
early observations concerning NO production was its dependence on L -arginine as a
substrate (Palmer et al., 1988). This observation prompted a series of studies ex-
amining the eect of L -arginine supplementation on vascular function. The admin-
istration of L -arginine has now been shown to enhance endothelium-derived NO
bioactivity in a number of disease states including coronary atherosclerosis (Quyy-
umi et al., 1997), variant angina (Egashira et al., 1996), hypercholesterolemia
(Drexler et al., 1991; Creager et al., 1992). These observations suggest that a de-
ciency of L -arginine may exist in patients with vascular disease. This does not agree
completely with the known enzymology of NOS as the KM for L -arginine is ap-
proximately 2:9 lM (Pollock et al., 1991), which is well below established intracel-
lular and plasma L -arginine concentrations (Harrison, 1997). One possible
explanation for this `àrginine paradox'' is that newly imported L -arginine may be a
better substrate for NO synthesis than the intracellular pool. This idea is supported
by observations that the L -arginine transporter is physically associated with NOS
and endothelial cells (McDonald et al., 1997). Another possible mechanism for the
arginine paradox relates to the presence of endogenous inhibitors of NOS. Once such
inhibitor is asymmetric-dimethyl arginine (ADMA) (Vallance et al., 1992). There are
observations that vascular disease is associated with increased plasma levels of
ADMA (Boger et al., 1998; Goonasekera et al., 1997), suggesting that the obser-
vations with L -arginine are due to displacement of ADMA. Thus, it is clear that
L -arginine treatment improves NO-mediated arterial relaxation, however the
mechanisms for such observations are still the subject of intense investigation.
4.1.5.2. Tetrahydrobiopterin. Another major cofactor for NOS is BH4 . This cofactor
is important for coupling the activation of oxygen with nitric oxide production
(Vasquez-Vivar et al., 1998; Bec et al., 1998; Xia et al., 1998b) and available evidence
indicates this cofactor improves NO responses in patients. For example, adminis-
tration of BH4 improves endothelial function in hypercholesterolemic patients
(Stroes et al., 1997), cigarette smokers (Higman et al., 1996; Heitzer et al., 2000), and
experimental models of diabetes (Pieper, 1997). These data suggest that intracellular
BH4 status may be limited in the setting of vascular disease.
4.1.5.3. LDL lowering therapy. Given that all the major theories of atherogenesis
involve LDL cholesterol (see above), it is not surprising that cholesterol lowering has
been investigated as a means to improve endothelial function. Moreover, lipid-low-
ering therapy has been shown to convincingly reduce the incidence of cardiovascular
events, and this occurs in the setting of modest reductions in atherosclerotic lesions
(Grundy, 1998). Such ndings prompt speculation that cholesterol-lowering therapy
improves the function of arteries. A number of studies have investigated this and they
are outlined in Table 8. Treatment with a variety of cholesterol-lowering therapies
has been shown to improve endothelial function over durations as short as one
month. In a very interesting study, Tamai et al. (1997) showed that LDL apheresis
improved endothelial function immediately in patients with relatively modest levels
of LDL cholesterol. These data underlie a specic interaction between LDL and
endothelial dysfunction that appears readily amenable to treatment (see Table 8).
4.1.5.4. Other treatments. A number of other interventions associated with reduced

cardiovascular risk also appear to translate into improved endothelial function. For
example, post-menopausal women treated with estrogen appear to have reduced
cardiovascular events (Stampfer et al., 1997), and consistent with this observation,
treatment of these same patients with estrogens appears to improve endothelial
function (Lieberman et al., 1994). Similarly, angiotensin converting enzyme inhibi-
tors are known to reduce coronary artery disease events (Pfeer et al., 1989), and
treatment of patients with quinapril for six months improves coronary artery en-
dothelial function (Mancini et al., 1996). Thus, there appears to be a close associ-
ation between interventions that improve cardiovascular outcome and the
improvement of endothelial function. Further studies will be required to identify the
precise mechanism for this association.
5. Summary
Atherosclerosis is an important source of morbidity and mortality in the de-

veloped world. Despite the fact that the association between LDL cholesterol and
atherosclerosis has been evident for at least three decades, our understanding of
exactly how LDL precipitates atherosclerosis is still in its infancy. At least three
working hypotheses of atherosclerosis are now nearing the stage where critical
evaluation is possible through a combination of basic science investigation and
murine models of atherosclerosis. As we move forward in our understanding of
this disease, eorts will be increasingly focused on the molecular mechanisms of
disease activation that precipitate the clinical manifestations of atherosclerosis
such as heart attack and stroke. Two candidates for such investigation involve the
events surrounding plaque activation and endothelial dysfunction. Further inves-
tigation in these elds should provide the necessary insight to develop the next
generation of interventions that will reduce the clinical manifestations of this

devastating disease.
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Molecular Aspects of Medicine 21 (2000) 99166

Atherosclerosis: from lesion formation to plaque

Keywords: Atherosclerosis; Inammation; Endothelium; Nitric oxide; Oxidation

0098-2997/00/$ - see front matter 2000 Published by Elsevier Science Ltd.

2.3. Response-to-injury hypothesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

Cardiovascular disease is the major source of morbidity and mortality in the

2.1. Epidemiology and risk factors

At the outset, one should understand that atherogenesis is a multifactorial

infarction, and 42% increase in stroke (MacMahon et al., 1990). Antihypertensive

2.2. Morphologic features of atherosclerosis

2.2.1. The normal artery

Fig. 1. Cross-section of a normal artery.

2.2.2. Gross morphology

2.3. Response-to-injury hypothesis

proliferation, Ross eventually proposed the ``response to injury'' hypothesis of

2.4. Oxidative modication hypothesis of atherosclerosis

2.4.1. LDL does not support foam cell formation

demonstrate a defect in the binding and degradation of modied LDL (Krieger,

2.4.2. Mechanisms of LDL oxidation

only that a-tocopherol and ubiquinol-10 possess important antioxidant activity

LOOH Men1 ! LOO Men H 1

Men LOOH ! LO OH Men1 2

mechanism, a single radical will produce damage to a number of subsequent PUFAs

2.4.2.7. Glycoxidation reactions. As discussed above, diabetes is a potent risk factor

produce may yield irreversible oxidation, or glycoxidation of fructoselysine to yield a

2.4.2.8. Peroxynitrite. Peroxynitrite is a well-characterized product of the radical

2.4.2.9. Myeloperoxidase. In the host-defense response, phagocytes produce super-

2.4.3. Proatherogenic actions of oxidized LDL

through LDL accumulation of lysophosphatidylcholine during oxidation (Steinbre-

2.4.4. Evidence to support the oxidative modication hypothesis

2.4.5. Antioxidant studies

2.4.6. Animal studies

The interpretation of animal antioxidant supplementation studies is not entirely

2.4.6.2. Probucol. Probucol is a cholesterol-lowering drug that is known to possess

similarly, however, probucol treatment provided an additional 48% reduction in the

2.4.7. Problems with the oxidative modication hypothesis

2.4.8. Clinical studies

2.4.9. Evidence from descriptive studies

In summary then, there is no clear consensus that antioxidant supplementation

2.5. The response-to-retention hypothesis of early atherogenesis

2.5.1. Evidence to support the response-to-retention hypothesis

lipoprotein lipase (LpL) enhances the adherence of LDL in vitro to proteoglycans

3. The concept of disease activity

3.1. The brous cap

For many years, end-organ ischemia was thought to result as a consequence of

3.2. Matrix degradation

Once a brous cap is formed over an atherosclerotic lesion, it is subject to a

metalloproteninases (TIMPs) (DeClerk et al., 1994). These proteins bind to matrix

of vascular homeostasis. Among the more important features of the endothelium is

4.1. Oxidative stress and impaired NO bioactivity

The exact mechanism producing abnormal bioactivity in the setting of vascular

4.1.1. Superoxide and NO bioactivity

such as hypercholesterolemia, diabetes, and hypertension are now noted to be as-

The relevance of this reaction on NO bioactivity is emphasized by experimental

CuZn SOD through chelation with diethyldithiocarbamate results in impaired

a greater extent than non-receptor-dependent stimuli (Lynch et al., 1997). This

4.1.2. Lipid peroxidation and NO bioactivity

4.1.3. Lipid-soluble antioxidants and NO bioactivity

4.1.3.1. a-Tocopherol and b-Carotene. The activity of a-tocopherol and b-carotene

4.1.4. Water-soluble antioxidants and NO bioactivity

a vasospastic angina, intracoronary glutathione improves NO bioactivity in response

4.1.4.2. Ascorbic acid. Ascorbic acid is a versatile antioxidant, it scavenges a wide

4.1.5. Other means of treating endothelial dysfunction